Bifunctional boronic compound complexing reagents and complexes

ABSTRACT

A reagent having the general formula of General Formula I: 
                 
 
wherein group R is an electrophilic or a nucleophilic moiety suitable for reaction of the reagent with a biologically active species; group R 2  is one of H and OH moieties; group R 3  is one of an alkyl and a methylene bearing an electronegative substituent; group Z is one of (CH 2 ) n  and CH 2 O(CH 2 CH 2 O) n     2   , and n is an integer of from 1 to 5, and n 2  is an integer of from 1 to 4; and each of group Z 2  and Z 3  are (CH 2 )CONH(CH 2 ) 3 CONH(CH 2 ) 2 . Also, a reagent having the general formula of General Formula II: 
                 
 
group R 2  is one of H and OH moieties; group Z is one of (CH 2 ) n  and CH 2 O(CH 2 CH 2 O) n     2   , and n is an integer of from 1 to 5, and n 2  is an integer of from 1 to 4; each of group Z 2  and group Z 3  include one of (CH 2 )CONH(CH 2 ) 3 CONH(CH 2 ) 2 ; and (CH 2 )CONH(CH 2 ); and group R is an electrophilic or a nucleophilic moiety suitable for reaction of the reagent with the biologically active species.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/651,007, filed Aug. 29, 2000, (U.S. Pat. No. 6,414,122), which is adivisional of U.S. patent application Ser. No. 09/222,468, filed Dec.29, 1998, (U.S. Pat. No. 6,156,884), which is a continuation-in-part ofU.S. patent application Ser. No 08/956,196, filed Oct. 22, 1997, (U.S.Pat. No. 5,877,297), which is a divisional application of U.S. patentapplication Ser. No. 08/691,930 filed Aug. 5, 1996, issued as U.S. Pat.No. 5,777,148, and a continuation-in-part of U.S. patent applicationSer. No. 08/956,194, filed Oct. 22, 1997, (U.S. Pat. No. 5,872,224),which is a divisional application of U.S. patent application Ser. No.08/689,283 filed date Aug. 5, 1996 (U.S. Pat. No. 5,837,878).

FIELD OF THE INVENTION

The present invention relates to the field of bioconjugate preparation,and more particularly, to a class of bifunctional boronic compoundcomplexing reagents useful for the conjugation of biologicalmacromolecules, and the method of making and using such reagents.

BACKGROUND OF THE INVENTION

Bioconjugation is a descriptive term for the joining of two or moredifferent molecular species by chemical or biological means, in which atleast one of the molecular species is a biological macromolecule. Thisincludes, but is not limited to, conjugation of proteins, peptides,polysaccharides, hormones, nucleic acids, liposomes and cells, with eachother or with any other molecular species that add useful properties,including, but not limited to, drugs, radionuclides, toxins, haptens,inhibitors, chromophores, fluorophores, ligands, etc. Immobilization ofbiological macromolecules is also considered a special case ofbioconjugation in which the macromolecule is conjugated, eitherreversibly or irreversibly, to an insoluble solid phase support.Bioconjugation is utilized extensively in biochemical, immunochemicaland molecuar biological research. Major applications of bioconjugationinclude; detection of gene probes, enzyme-linked immuno solid-phaseassay, monoclonal antibody drug targeting and medical imaging.

Bioconjugates are generally classified as either direct or indirectconjugates. Direct conjugates encompass those in which two or morecomponents are joined by direct covalent chemical linkages.Alternatively, indirect conjugates encompass those in which two or morecomponents are joined via an intermediary complex involving a biologicalmacromolecule. The system described herein is the first to enable theformation of indirect conjugates without dependence upon an intermediarybiological macromolecule.

Avidin-Biotin System

Although numerous methods of indirect bioconjugate preparation have beendescribed, a significant number of those reported in the literature havebeen prepared by exploiting the Avidin-Biotin system, in which, thebinding specificity of the protein Avidin (purified from egg white), orStreptavidin (purified from the bacterium Streptomyces avidinii), towardthe cofactor Biotin (vitamin H) is utilized to bridge an Avidinconjugated macromolecule with a biotinylated macromolecule. Both Avidinand Streptavidin possess four Biotin binding sites of very high affinity(K_(a)=10¹⁵ M⁻¹).

The Avidin-Biotin system has been utilized extensively for enzyme-linkedimmuno solid-phase assay (ELISA), in which an enzyme-Avidin conjugate(useful for detection by reaction with the enzyme's substrate to afforda colored or chemiluminescent product) is employed to detect thepresence of a biotinylated antibody, after first binding the antibody toan immobilized antigen or hapten. Applications of the Avidin-Biotinsystem number in the hundreds, and have recently been reviewed (Wilchek,M. and Bayer, E. A., (1990) Methods in Enzymology, 184).

Although utilized extensively, several limitations are known to beassociated with the Avidin-Biotin system, which include nonspecificbinding generally attributed to the basicity of the Avidin molecule,nonspecific binding attributed to the presence of carbohydrate residueson the Avidin molecule, and background interference associated with thepresence of endogenous Biotin, which is ubiquitous in both eukaryoticand prokaryotic cells.

Digoxigenin Anti-Digoxigenin System

An alternative indirect bioconjugation system designed to overcome someof the limitations associated with the Avidin-Biotin system has recentlybeen developed for the detection of gene probes by ELISA (Kessler, C.,Hôltke, H.-J., Seibl, R., Burg, J. and Mühlegger, K., (1990) Biol. Chem.Hoppe-Seyler, 371, 917-965). This system involves the use of the steroidhapten Digoxigenin, an alkaloid occuring exclusively in Digitalisplants, and Fab fragments derived from polyclonal sheep antibodiesagainst Digoxigenin (anti-Digoxigenin). The high specificity of thevarious anti-Digoxigenin antibodies affords low backgrounds andeliminates the non-specific binding observed in Avidin-Biotin systems.Digoxigenin-labeled DNA and RNA probes can detect single-copy sequencesin human genomic Southern blots. The development of the Digoxigeninanti-Digoxigenin system has recently been reviewed (Kessler, C. (1990)in Advances in Mutagenesis Research (Obe, G. ed.) pp. 105-152,Springer-Verlag, Berlin/Heidelberg). The Digoxigenin anti-Digoxigeninsystem is the most recent representative of several hapten-antibodysystems now utilized extensively for bioconjugation.

Immobilized Phenylboronates

Phenylboronic acids are known to interact with a wide range of polarmolecules having certain requisite functionalities. Complexes of varyingstability, involving 1,2-diols, 1,3-diols, 1,2-hydroxy acids,1,3-hydroxy acids, 1,2-hydroxylamines, 1,3-hydroxylamines, 1,2-diketonesand 1,3-diketones, are known to form with either neutral phenylboronicacid or phenylboronate anion. Consequently, immobilized phenylboronicacids have been exploited as chromatographic supports to selectivelyretain, from diverse biological samples, those molecular species havingthe requisite functionalities. Many important biological moleculesincluding carbohydrates, catecholamines, prostaglandins,ribonucleosides, and steroids contain the requisite functionalities, andhave been either analyzed or purified in this manner. The use ofphenylboronic acid chromatographic media for the isolation andseparation of biological molecules has been discussed in several reviews(Singhal, R. P. and DeSilva, S. S. M. (1992) Adv. Chromatog., 31,293-335; Mazzeo, J. R. and Krull, I. S. (1989) BioChromatog., 4,124-130; and Bergold, A. and Scouten, W. H. (1983) in Solid PhaseBiochemistry (Scouten, W. H. ed.) pp. 149-187, John Wiley & Sons, NewYork).

Phenylboronic acid, like boric acid, is a Lewis acid, and ionizes not bydirect deprotonation, but by hydration to give the tetrahedralphenylboronate anion (pK_(a)=8.86). Phenylboronic acid is three times asstrong an acid as boric acid. Ionization of phenylboronic acid is animportant factor in complex formation, in that, upon ionization, boronchanges from trigonal coordination (having average bond angles of 120°and average bond lengths of 1.37 angstroms) to the tetrahedralcoordinated anion (having average bond angles of 109° and average bondlengths of 1.48 angstroms).

Molecular species having cis or coaxial 1,2-diol and 1,3-diolfunctionalities, and particularly carbohydrates, are known to complexwith immobilized phenylboronate anion, to form cyclic esters underalkaline aqueous conditions (Lorand, J. P. and Edwards, J. O. (1959) J.Org. Chem., 24, 769).

Acidification of 1,2-diol and 1,3-diol complexes to neutral pH is knowto release the diol containing species, presumably due to hydrolysis ofthe cyclic ester. Coplanar aromatic 1,3-diols, like1,8-dihydroxynaphthalene, are known to complex even under acidicconditions due to the hydrolytic stability of six-membered cyclicboronic acid esters (Sienkiewicz, P. A. and Roberts, D. C. (1980) J.Inorg. Nucl. Chem., 42, 1559-1571). Molecular species having pendant1,2-hydroxylamine, 1,3-hydroxylamine, 1,2-hydroxyamide,1,3-hydroxyamide, 1,2-hydroxyoxime and 1,3-hydroxyoxime functionalitiesare also known to reversibly complex with phenylboronic acid underalkaline aqueous conditions similar to those associated with theretention of diol containing species (Tanner, D. W. and Bruice, T. C.(1967) J. Amer. Chem. Soc., 89, 6954).

Phenylboronate Bioconjugates

Ortho-substituted acetamidophenylboronic acids have been proposed aspotential linkers for selective bioconjugation via the vicinal diolmoieties of the carbohydrate residues associated with glycoproteins(Cai, S. X. and Keana, J. F. W. (1991) Bioconjugate Chem., 2, 317-322).Phenylboronic acid bioconjugates derived from3-isothiocyanatophenylboronic acid have been successfully utilized forappending radioactive technetium dioxime complexes to monoclonalantibodies for use in medical imaging (Linder, K. E., Wen, M. D.,Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D. andEckelman, W. C. (1991) Bioconjugate Chem., 2, 160-170; Linder, K. E.,Wen, M. D., Nowotnik, D. P., Ramalingam, K., Sharkey, R. M., Yost, F.,Narra, R. K. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 407-414).

3-Aminophenylboronic acid has been covalently appended to proteins by avariety of chemical methods and the resulting phenylboronic acidbioconjugates tested for their binding of D-sorbitol, D-mannose andglycated hemoglobin (GHb). The interactions proved to be reversible andof very low affinity rendering the bioconjugates of very limitedpractical use. Similarly, an alkaline phosphatase phenylboronic acidbioconjugate used in an attempted enzyme-linked assay for the detectionof GHb failed to detect the presence of glycated protein (Frantzen, F.,Grimsrud, K., Heggli, D. and Sundrehagen, E. (1995) Journal ofChromatography B, 670, 37-45).

Phenylboronic Acid Complexing Reagents

A novel class of phenylboronic acid reagents and phenylboronic acidcomplexing reagents have been developed for conjugating biologicallyactive species by exploiting indirect bioconjugation through areversible boron complex. These reagents and associated conjugates maybe used in a manner analogous to Avidin-Biotin andDigoxigenin-anti-Digoxigenin systems. However, unlike the Avidin-Biotinand Digoxigenin-anti-Digoxigenin systems, wherein the viability of thebiological macromolecule must be maintained to preserve requisitebinding properties, the bioconjugate formed through the boron complex isgenerally insensitive to significant variations in ionic strength,temperature, the presence of organic solvents, and the presence ofchaotropic agents (protein denaturants).

Phenylboronic acid reagents, phenylboronic acid complexing reagents,their conjugates and bioconjugates, as well as methods for theirpreparation and use are the subject of U.S. Pat. Nos. 5,594,111,5,623,055, 5,668,258, 5,648,470, 5,594,151, 5,668,257, 5,677,431,5,688,928, 5,744,627, 5,777,148, 5,831,045, 5,831,046 and 5,837,878.

SUMMARY OF THE INVENTION

The present invention relates to a novel class of bifunctional boroniccompound complexing reagents useful for the preparation ofbioconjugates, and the method of making and using such reagents. In oneembodiment, the boron compound is phenylboronic acid, or derivativesthereof, which complex with the complexing reagents of the presentinvention. In a second embodiment, the boron compound is phenyldiboronicacid, or derivatives thereof, which complex with the complexing reagentsof the present invention. Unless otherwise noted, the phrasebifunctional boronic compound complexing reagent is used herein toinclude the broader class of boron compound complexing reagents whichcomplex with boron compounds, and the phrase phenylboronic acid is usedherein to include the broader class of boron compounds which complexwith the boron compound complexing reagents, including bifunctionalboronic compound complexing reagents. In the present invention, in theplace of prior art Avidin-Biotin and Digoxigenin anti-Digoxigeninsystems, bifunctional boronic compound complexing reagents comprised oftwo boron compound complexing moieties can be utilized in conjunctionwith the boron compound, such as phenylboronic acid reagents (many ofwhich are known in the prior art) to facilitate chemical conjugation andprepare bioconjugates without the use of intermediary biologicalmacromolecules. Bioconjugate preparation often involves the conjugationof several components including, but not limited to, proteins, peptides,polysaccharides, hormones, nucleic acids, liposomes and cells, with eachother or with any other molecular species that add useful properties,including, but not limited to, drugs, radionuclides, toxins, haptens,inhibitors, fluorophores, ligands, solid-phase supports, and boroncompound complexing reagents conjugates. These various componentsutilized in bioconjugate preparation will collectively and individuallybe termed biologically active species or bioactive species.

Two alternative methods for the preparation of bioconjugates derivedfrom bifunctional boronic compound complexing reagents are disclosedbelow. In the first method, which is comprised of three steps, a reagenthaving putative boronic compound complexing moieties is first prepared,and then converted into a boronic compound complexing conjugate prior toreaction with a phenylboronic acid conjugate to afford a bioconjugate.In the second method, which is comprised of two steps, a boroniccompound complexing conjugate is prepared in a single step, and thenreacted with a phenylboronic acid conjugate to afford a bioconjugate.

Three-Step Method of Bioconjugate Preparation

Reagents suitable for the modification of a bioactive species for thepurpose of incorporating a bifunctional boronic compound complexingmoiety for subsequent conjugation to a different (or the same) bioactivespecies having pendant phenylboronic acid moieties are of GeneralFormula I,

wherein group R is an electrophilic or nucleophilic moiety suitable forreaction of the putative bifunctional boronic compound complexingreagent with a bioactive species, wherein group R₂ is selected from oneof H and OH moieties, and wherein group R₃ is selected from one of analkyl (e.g., methyl, ethyl, etc.) and a methylene bearing anelectronegative substituent.

Group Z is a spacer selected from (CH₂)_(n) and CH₂O(CH₂CH₂O)_(n) ₂ ,wherein n is an integer of from 1 to 5, and wherein n₂ is an integer offrom 1 to 4. Each of group Z₂ and Z₃ is a spacer selected from CH₂Ar,CH₂CONHCH₂Ar, CH₂CONH(CH₂)_(n) ₃ CO—NHCH₂Ar, and (CH₂)_(n) ₄NHCO(CH₂)_(n) ₅ CONHCH₂Ar, wherein the group Ar represents the aromaticring in the reagent of General Formula I to which the spacer Z₂ or Z₃ isappended, wherein n₃ is an integer of from 1 to 5, wherein n₄ is aninteger selected from one of 2 and 3, and wherein n₅ is an integer offrom 1 to 4. It is to be appreciated that, for a given reagent ofGeneral Formula I, spacers Z₂ and Z₃ need not be the same moiety.

Reaction of a reagent of General Formula I with a bioactive speciesaffords a conjugate having pendant putative bifunctional boroniccompound complexing moieties (one or more) of General Formula II,

wherein groups R₂, R₃, Z, Z₂ and Z₃ are as were previously defined, andwherein the symbol labeled BAS represents a biologically active species(or bioactive species) that may or may not contain a portion of areactive moiety (which may itself have a spacer) used to attach thebioactive species to the reagent.

It will be appreciated that, in many embodiments, several identicalreagents of General Formula I will react with a single BAS molecule. Forexample, if the BAS is a protein, many bifunctional boronic compoundcomplexing reagents will react with the protein, each reacting at one ofthe several sites on the protein which are reactive with the R group.

The conjugate of General Formula II may be further reacted withhydroxylamine (NH₂OH) by amidation of the benzoic acid ester moiety toafford a class of bifunctional boronic compound complexing conjugate,e.g., conjugate with one or more pendant bifunctional boronic compoundcomplexing moieties of General Formula III,

wherein groups R₂, Z, Z₂, Z₃ and BAS are as were previously defined.

Phenylboronic acid reagents, many of which are known in the prior art,as well as those described in greater detail in my copendingapplication, titled “Phenyldiboronic Acid Reagents and Complexes”, filedAug. 21, 1998, U.S. patent application Ser. No. 09/138,105, (U.S. Pat.No. 6,075,126), which is incorporated herein by reference, may beappended to a biologically active species to afford a conjugate havingpendant phenylboronic acid moieties (one or more) of the general formulaof General Formula IV,

wherein group R₄ is selected form one of H and B(OH)₂ moieties, andwherein the symbol labeled BAS* represents a second bioactive species,that may include a linker portion and that may differ from the bioactivespecies labeled BAS. The BAS* may also include a portion of a reactivemoiety used to attach the bioactive species to the phenylboronic acidreagent.

A conjugate of General Formula III, with at least one biologicallyactive species and having pendent bifunctional boronic compoundcomplexing moieties (one or more), may be complexed with a conjugate ofGeneral Formula IV, prepared from a second bioactive species BAS* andhaving pendant phenylboronic acid moieties (one or more), to afford abioconjugate of General Formula V,

wherein the symbols labeled BAS and BAS*, and groups R₂, R₄, Z, Z₂ andZ₃, are as were previously defined. In this manner, biologicalmacromolecules may be conjugated to one another or with otherfunctionalities that impart useful properties.

Two-Step Method of Bioconjugate Preparation

Alternatively, a second class of reagents suitable for the modificationof a bioactive species for the purpose of incorporating a bifunctionalboronic compound complexing moiety for subsequent conjugation to adifferent (or the same) bioactive species having pendant phenylboronicacid moieties are of General Formula VI,

wherein group R₁ is a reactive electrophilic or nucleophilic moietysuitable for reaction of the putative bifunctional boronic compoundcomplexing reagent with a bioactive species, and wherein group R₂ isselected from one of H and OH moieties. Group Z is a spacer selectedfrom (CH₂)_(n) and CH₂O(CH₂CH₂O)_(n) ₂ , wherein n is an integer of from1 to 5, and wherein n₂ is an integer of from 1 to 4. Each of group Z₂and group Z₃ is a spacer selected from CH₂Ar, CH₂CONHCH₂Ar,CH₂CONH(CH₂)_(n) ₃ CONHCH₂Ar, and (CH₂)_(n) ₄ NHCO(CH₂)_(n) ₅ CONHCH₂Ar,wherein the group Ar represents the aromatic ring in the reagent ofGeneral Formula VI to which the spacer Z₂ or Z₃ is appended, wherein n₃is an integer of from 1 to 5, wherein n₄ is an integer selected from oneof 2 and 3, and wherein n₅ is an integer of from 1 to 4. It is to beappreciated that, for a given reagent of General Formula VI, spacers Z₂and Z₃ need not be the same moiety.

Reaction of a reagent of General Formula VI with a bioactive speciesaffords a conjugate having pendant boronic compound complexing moieties(one or more) of General Formula VII,

wherein groups R₂, Z, Z_(2,) and Z₃ are as were previously defined, andwherein the symbol BAS represents the bioactive species that may or maynot contain a portion of a reactive moiety used to attach the bioactivespecies.

In a manner indistinguishable from that described above for thethree-step method, a conjugate of General Formula VII (which iscomparable to a conjugate of General Formula III), may be complexed witha conjugate of General Formula IV, to afford a bioconjugate of GeneralFormula V,

wherein the symbols labeled BAS and BAS*, and groups R₂, R₄, Z, Z₂ andZ₃ are as were previously defined. In this manner, biologicalmacromolecules may be conjugated to one another or with otherfunctionalities that impart useful properties.

Bioconjugates of General Formula V, whether formed, for example, inaccordance with either the three-step method or the two-step methoddescribed above, may be prepared in buffered aqueous solution or organicsolvents. The bioconjugate is formed within a few minutes over a rangeof temperatures from about 4° C. to 70° C. The stability of thebioconjugate in aqueous solution at a given pH and temperature issignificantly influenced by substituent group R₂. Bioconjugates ofGeneral Formula V, wherein group R₄ is H, are stable in aqueoussolutions of approximate pH greater than 3.5 and less than 12.5.Bioconjugates of General Formula V, wherein group R₂ is OH, are stablein aqueous solutions of approximate pH greater than 1.5 and less than12.5.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the utilization of putative bifunctional boroniccompound complexing reagents of General Formula I to prepare conjugatesof General Formula III, which may, in turn, be utilized to preparebioconjugates of General Formula V.

FIG. 2 illustrates the utilization of putative bifunctional boroniccompound complexing reagents of General Formula VI to prepare conjugatesof General Formula VII, which may, in turn, be utilized to preparebioconjugates of General Formula V.

FIG. 3 illustrates the utilization of a bifunctional boronic compoundcomplexing conjugate of either General Formula III or General FormulaVII to prepare bioconjugates of General Formula VIII.

FIG. 4 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group Z is (CH₂)_(n), wherein n is from 1 to 5,wherein group Z₂ and group Z₃ are each CH₂, and wherein group R₃ is analkyl (e.g., methyl, ethyl, etc.) moiety.

FIG. 5 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group R is a reactive electrophilicN-hydroxysuccinimidyl ester moiety, wherein group Z is (CH₂)₃, whereingroup Z₂ and group Z₃ are each CH₂, and wherein group R₃ is an alkyl(e.g., methyl, ethyl, etc.) moiety.

FIG. 6 summarizes a method for the preparation of reagents of GeneralFormula I derived from diethylenetriamine, wherein group R is a reactiveelectrophilic or nucleophilic moiety, wherein group R₃ is selected fromone of an alkyl (e.g., methyl, ethyl, etc.) and a methylene bearing anelectronegative substituent, wherein group Z is (CH₂)_(n), wherein n isfrom 1 to 5, and wherein group Z₂ and group Z₃ are eachCH₂CH₂NHCO(CH₂)₃CONHCH₂Ar, wherein the group Ar represents the aromaticring to which the spacer Z₂ is appended.

FIG. 7 summarizes a method for the preparation of reagents of GeneralFormula VI derived from diethylenetriamine, wherein group R₁ is areactive electrophilic or nucleophilic moiety, wherein group Z is(CH₂)_(n), wherein n is from 1 to 5, and wherein group Z₂ and group Z₃are each CH₂CH₂NHCO(CH₂)₃CO—NHCH₂Ar, wherein the group Ar represents thearomatic ring to which the spacer Z₂ or Z₃ is appended.

FIG. 8 summarizes a method for the preparation of reagents of GeneralFormula I derived from diethylenetriamine, wherein group R is a reactiveelectrophilic N-hydroxysuccinimidyl ester moiety, wherein group R₃ isselected from one of an alkyl (e.g., methyl, ethyl, etc.) and amethylene bearing an electronegative substituent, wherein group Z is(CH₂)₂, and wherein group Z₂ and group Z₃ are eachCH₂CH₂NHCO(CH₂)₃CONHCH₂Ar, wherein the group Ar represents the aromaticring to which the spacer Z₂ or Z₃ is appended.

FIG. 9 summarizes a method for the preparation of reagents of GeneralFormula I derived from iminodiacetic acid, wherein group R is a reactiveelectrophilic N-hydroxysuccinimidyl ester moiety, wherein group R₃ isselected from one of an alkyl (e.g., methyl, ethyl, etc.) and amethylene bearing an electronegative substituent, wherein group Z is(CH₂)₃, and wherein group Z₂ and group Z₃ are each CH₂CONHCH₂Ar, whereinthe group Ar represents the aromatic ring to which the spacer Z₂ or Z₃is appended.

FIG. 10 summarizes a method for the preparation of reagents of GeneralFormula VI derived from iminodiacetic acid, wherein group R₁ is areactive nucleophilic hydrazide moiety, wherein group Z is (CH₂)₃, andwherein group Z₂ and group Z₃ are each CH₂CONHCH₂Ar, wherein the groupAr represents the aromatic ring to which the spacer Z₂ or Z₃ isappended.

FIG. 11 summarizes a method for the preparation of reagents of GeneralFormula I derived from iminoacetic acid, wherein group R is a reactiveelectrophilic or nucleophilic moiety, wherein group R₃ is selected fromone of an alkyl (e.g., methyl, ethyl, etc.) and a methylene bearing anelectronegative substituent, wherein group Z is (CH₂)_(n), wherein n isfrom 1 to 5, wherein group Z₂ and group Z₃ are each CH₂CONH(CH₂)_(n) ₃CONHCH₂Ar, wherein n₃ is from 1 to 5, and wherein the group Arrepresents the aromatic ring to which the spacer Z₂ or Z₃ is appended.

DETAILED DESCRIPTION OF THE INVENTION

The three-step method which utilizes reagents of General Formula I forthe preparation of bioconjugates is summarized in FIG. 1. Initially, areagent of General Formula I is selected that is comprised of anappropriate electrophilic or nucleophilic group R suitable for reactionwith the desired biologically active species.

Group R is an electrophilic or nucleophilic moiety suitable for reactionof the putative bifunctional boronic compound complexing reagent with abioactive species, and is preferably selected from, but not limited to,one of acrylamide, bromo, dithiopyridyl, bromoacetamide, chloro,chloroacetamide, hydrazide, N-hydroxysuccinimidyl ester,N-hydroxysulfosuccinimidyl ester, imidate ester, imidazolide, iodo,iodoacetamide, maleimide, amino and thiol moieties.

Group R₂ is selected from one of H and OH moieties.

Group R₃ is selected from one of an alkyl (e.g., methyl, ethyl, etc.)and a methylene bearing an electronegative substituent. Anelectronegative substituent is a substituent with a negative dipolemoment, e.g., CN, COOH, etc. Group R₃ is preferably selected from one ofCH₃, CH₂CH₃, CH₂CN, CH₂COOH, CH₂CONH₂ and CH₂OCH₃.

Group Z is a spacer selected from one of (CH₂)_(n) and CH₂O(CH₂CH₂O)_(n)₂ , wherein n is an integer of from 1 to 5, and wherein n₂ is an integerof from 1 to 4.

Each of group Z₂ and group Z₃ is a spacer selected from one of CH₂Ar,CH₂CONHCH₂Ar, CH₂CONH(CH₂)_(n) ₃ CONHCH₂Ar and (CH2)_(n) ₄ NHCO(CH₂)_(n)₅ CO—NHCH₂Ar, wherein the group Ar represents the aromatic ring in thereagent of General Formula I to which the spacer Z₂ or Z₃ is appended,wherein n₃ is an integer of from 1 to 5, wherein n₄ is an integerselected from one of 2 and 3, and wherein n₅ is an integer of from 1 to4. It is to be appreciated that, for a given reagent of General FormulaI, spacers Z₂ and Z₃ need not be the same moiety.

The next step in a three-step method for the preparation ofbioconjugates is to condense the appropriate reagent with the bioactivespecies to yield a conjugate of General Formula II,

wherein groups R₂, R₃, Z, Z₂ and Z₃ are as were previously defined, andwherein the symbol BAS represents a biologically active species whichmay or may not contain a portion of a reactive moiety used to attach thebiologically active species to the reagent.

Next, the conjugate of the general formula of General Formula II isreacted with an excess of an aqueous solution of hydroxylamine to afforda conjugate of General Formula III,

wherein groups Z, Z_(2,) Z_(3,) R₂ and BAS are as were previouslydefined.

Aqueous solutions of hydroxylamine (NH₂OH) suitable for use in theaforementioned reaction are in the concentration range 0.1 to 1 molar,and are prepared by addition of solid sodium hydroxide to an aqueoussolution of hydroxylamine hydrochloride to obtain an approximate pH of10. The reactivity of hydroxylamine toward the benzoate esters ofGeneral Formula II is determined by the choice of group R₃. When R₃ (inGeneral Formula II) is an alkyl group, reaction overnight with 0.5 Mhydroxylamine at room temperature may be required to insure completeconversion of the benzoate ester to the corresponding hydroxamic acid.However, when R₃ is a methylene group bearing an electronegativesubstituent, and is preferably the cyanomethyl group (CH₂CN),quantitative conversion of the benzoate ester to the correspondinghydroxamic acid is complete within 2 hours with 0.1 M hydroxylamine at4° C.

The bifunctional boronic compound complexing conjugate of GeneralFormula III is next complexed with a phenylboronic acid conjugate havingthe general formula of General Formula IV:

wherein group R₄ is selected from one of H and B(OH)₂ moieties, andwherein the symbol labeled BAS* represents a second biologically activespecies, that may include a linker portion and differ from thebiologically active species labeled BAS of the complexing reagent. TheBAS* may also include a portion of a reactive moiety used to attach thebioactive species to the phenylboronic acid reagent. The complexationreaction yields a bioconjugate of the general formula of General FormulaV,

wherein groups Z, Z₂, Z₃, R₂, R₄, BAS and BAS* are as were previouslydefined.

Alternatively, a two-step method which utilizes reagents of GeneralFormula VI for the preparation of bioconjugates is summarized in FIG. 2.Initially, a reagent of General Formula VI is selected that is comprisedof an appropriate reactive electrophilic or nucleophilic group R₁suitable for reaction with the desired biologically active species.

Group R₁ is an electrophilic or nucleophilic moiety suitable forreaction of the bifunctional boronic compound complexing reagent with abioactive species. The presence of the hydroxamic acid moiety in generalFormula VI may limit the potential scope of reactive electrophilic ornucleophilic moieties suitable for reaction of the reagent with abioactive species, owing to the cross-reactivity known to occur betweenotherwise suitable moieties and the hydroxamic acid moiety.Consequently, in one embodiment group R₁ is preferably selected from,but not limited to, one of acrylamide, amino, dithiopyridyl, hydrazide,imidate ester, maleimide, and thiol moieties.

Group R₂ is selected from one of H and OH moieties.

Group Z is a spacer selected from one of (CH₂)_(n) and CH₂O(CH₂CH₂O)_(n)₂ , wherein n is an integer of from 1 to 5, and wherein n₂ is an integerof from 1 to 4.

Each of group Z₂ and group Z₃ is a spacer selected from one of CH₂Ar,CH₂CONHCH₂Ar, CH₂CONH(CH₂)_(n) ₃ CONHCH₂Ar and (CH₂)_(n) ₄ NHCO(CH₂)_(n)₅ CO—NHCH₂Ar, wherein the group Ar represents the aromatic ring in thereagent of General Formula VI to which the spacer Z₂ or Z₃ is appended,wherein n₃ is an integer of from 1 to 5, wherein n₄ is an integerselected from one of 2 and 3, and wherein n₅ is an integer of from 1 to4.

The next step in a two-step process for the preparation of bioconjugatesis to condense the appropriate reagent with the bioactive species toyield a conjugate of the general formula of General Formula VII,

wherein groups R₂, Z, Z₂ and Z₃ are as were previously defined, andwherein the symbol labeled BAS represents a biologically active specieswhich may or may not contain a portion of a reactive moiety used toattach the biologically active species to the reagent.

Finally, the boronic compound complexing conjugate of General FormulaVII is complexed with a phenylboronic acid conjugate of General FormulaIV to afford a bioconjugate of the general formula of General Formula V,

wherein group R₄ is selected from one of H and B(OH)₂ moieties. Thesymbol labeled BAS* represents a second biologically active species thatmay or may not contain a portion of a reactive moiety used to attach thebiologically active species to the reagent. Groups R₂, Z, Z₂, Z₃, andBAS are as were previously defined.

Bioconjugates Comprised of Three Bioactive Species

As illustrated in FIG. 3, the presence of two boronic compoundcomplexing moieties in conjugates of General Formulas III and VII,enable the potential for the preparation of bioconjugates comprised of asingle bifunctional boronic compound complexing conjugate and twophenylboronic acid conjugates of the general formula of General FormulaVIII,

wherein groups R₂, R₄, Z, Z₂, Z₃, BAS and BAS* are as were previouslydefined.

Preparation of Bioconjugates of General Formulas V and VIII

Bioconjugates of General Formulas V and VIII may be prepared in bufferedaqueous solutions or organic solvents. Preferred buffers includeacetate, citrate, phosphate, carbonate and diglycine. Borate buffersshould be avoided due to their ability to complex with the boroniccompound complexing moiety. Trishydroxymethylamino-methane,β-hydroxyamine and β-hydroxyacid buffers should also be avoided due totheir ability to complex with the phenylboronic acid. The bioconjugateis formed within a few minutes over a range of temperatures of fromabout 4° C. to 70° C. The bio-conjugation reaction (phenylboronic acidcomplexation) is insensitive to significant variations in ionic strengthover the range 0.01 to 1 M, the presence of organic solvents includingacetonitrile, methanol, ethanol, isopropanol, butanol,N,N-dimethylformamide and dimethylsulfoxide, the presence of detergents,and the presence of chaotropic agents (protein denaturants) includingurea, guanidine hydrochloride, guanidine thiocyanate and formamide,which are incompatible with prior art indirect labeling systems whereinthe structure of a biological macromolecule must be maintained topreserve requisite binding properties. Once formed, the bioconjugatesare stable upon removal of water, and can be lyophilized for storage.

The stability of the bioconjugate in aqueous solutions at a given pH isdetermined to some extent by substituent group R₂. Phenylboronic acidcomplexes of General Formulas V and VIII, wherein group R₂ is H, arestable in buffered aqueous solutions over the approximate pH range 3.5to 12.5. Phenylboronic acid complexes of General Formulas V and VIII,wherein group R₂ is OH, are stable in buffered aqueous solutions overthe approximate pH range 1.5 to 12.5.

Synthesis of Reagents of General Formulas I and VI

Reagents of General Formulas I and VI are prepared from syntheticintermediates that are also utilized in the syntheses of the variousmonofunctional boronic acid complexing reagents which are described inU.S. Pat. Nos. 5,777,148, 5,744,627, and 5,837,878, as well as ourcopending applications: U.S. patent application Ser. No. 08/689,341(U.S. Pat. No. 5,837,878), titled Boronic Compound Complexing Reagentsand Complexes, filed Aug. 5, 1996; U.S. patent application Ser. No.08/691,929 (U.S. Pat. No. 5,777,148), titled Boronic Compound ComplexingReagents and Highly Stable Complexes, filed Aug. 5, 1996; U.S. patentapplication Ser. No. 08/689,283 (U.S. Pat. No. 5,837,878), titledBoronic Compound Complexing Reagents and Highly Stable Complexes, filedAug. 5, 1996; U.S. patent application Ser. No. 08/691,930 (U.S. Pat. No.5,837,878), titled Boronic Compound Complexing Reagents and Complexes,filed Aug. 5, 1996, which are incorporated herein by reference.

In particular, the aforementioned patents and copending applicationsdetail the syntheses of the following compounds which are usefulsynthetic intermediates: methyl 4-aminomethyl-2-hydroxybenzoatehydrochloride, methyl 4-bromomethyl-2-hydroxy-benzoate, methyl4-aminomethyl-2,6-dihydroxybenzoate hydrochloride, methyl4-bromomethyl-2,6-dihydroxybenzoate, cyanomethyl4-aminomethyl-2-hydroxybenzoate hydrochloride,ethyl(6-aminohexanoyl)aminomethyl-2-hydroxybenzoate trifluoroacetate,methyl(6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate hydrochloride,methyl 4-succinylaminomethyl-2-hydroxybenzoate succinimidyl ester,methyl(6-amino-hexanoyl)aminomethyl-2-hydroxybenzoate hydrochloride,cyanomethyl 4-glutaryl-aminomethyl-2-hydroxybenzoate succinimidyl ester,and methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate succinimidylester.

The preparation of bifunctional boronic compound complexing reagents ofGeneral Formulas I and VI are summarized in FIGS. 4 through 10.

FIG. 4 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group Z is (CH₂)_(n), wherein groups Z₂ and Z₃ areeach CH₂Ar, wherein the group Ar represents the aromatic ring to whichthe spacer Z₂ is appended, and wherein group R₃ is an alkyl (e.g.,methyl, ethyl, etc.) moiety. Condensation of the benzylamine adduct withthe benzylbromide adduct affords the secondary amine intermediate, whichis isolated as the corresponding hydrochloride salt. It is to beappreciated that different moieties for spacers Z₂ and Z₃ may beformulated at this step by modifying the condensation reactants.

Neutralization of the amine hydrochloride produced by the condensationreaction and subsequent reaction with di-tert-butyldicarbonate affordsthe tert-butyloxycarbonyl (BOC) protected secondary amine. Alkylation ofthe phenolic hydroxyls associated with the BOC protected secondary aminewith benzylbromide next affords the dibenzyl protected BOC secondaryamine intermediate, which is reacted with anhydrous hydrogen chloridegas to effect removal of the BOC protecting group. The dibenzylprotected intermediate is next condensed with a carboxylic acid of thegeneral formula R′(CH₂)_(n) COOH, wherein group R′ is selected from oneof a reactive electrophilic or nucleophilic moiety, and a protectedreactive electrophilic or nucleophilic moiety having a protecting groupwhich is subject to removal in the final step of the synthesis, andwherein n is from 1 to 5. The carboxylic acid is activated by reactionwith isobutyl chloroformate prior to condensation with the dibenzylprotected intermediate. The reaction affords the dibenzyl protectedpenultimate product. Finally, the benzyl protecting groups, as well asany protecting groups associated with group R′, are removed byhydrogenation over palladium on carbon to afford a reagent of GeneralFormula I.

FIG. 5 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group R is a reactive electrophilicN-hydroxysuccinimidyl ester moiety, wherein group Z is (CH₂)₃, whereingroups Z₂ and Z₃ are each CH₂Ar, wherein the group Ar represents thearomatic ring to which the spacer Z₂ or Z₃ is appended, and whereingroup R₃ is an alkyl (e.g., methyl, ethyl, etc.) moiety. In the initialstep of the synthesis, the secondary amine intermediate is prepared asdescribed above with reference to FIG. 4. Condensation of the secondaryamine intermediate with glutaric anhydride next affords thecorresponding carboxylic acid intermediate, which is furtherfunctionalized by reaction with 1,3-dicyclohexylcarbodiimide andN-hydroxysuccinimide to afford a reagent of General Formula I, wherein Ris a reactive electrophilic N-hydroxysuccinimidyl ester moiety.

FIG. 6 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group R is a reactive electrophilic or nucleophilicmoiety, wherein group R₃ is selected from one of an alkyl (e.g., methyl,ethyl, etc.) and a methylene bearing an electronegative substituent,wherein group Z is (CH₂)_(n), wherein n is from 1 to 5, and whereingroups Z₂ and Z₃ are each CH₂CH₂NHCO(CH₂)₃CO—NHCH₂Ar, wherein the groupAr represents the aromatic ring to which the spacer Z₂ or Z₃ isappended. Condensation of diethylenetriamine with two equivalents of areagent selected from one of methyl, ethyl and cyanomethyl4-glutarylaminomethyl-2-hydroxybenzoate succinimidyl ester affords thesecondary amine intermediate, which is isolated as the correspondingN-hydroxysuccinimide salt. To afford different spacers for each of Z₂and Z₃, one equivalent of a first succinimidyl ester may be condensedwith the diethylenetriamine followed by condensation with one equivalentof a second succinimidyl ester different from the first ester. Afterneutralization of the N-hydroxysuccinimide salt, the secondary amineintermediate is condensed with a carboxylic acid of the general formulaR(CH₂)_(n) COOH, wherein group R is a reactive electrophilic ornucleophilic moiety, and wherein n is from 1 to 5. The carboxylic acidis first activated by reaction with 1,3-dicyclohexylcarbodiimide andN-hydroxysuccinimide, and affords upon reaction with the secondary amineintermediate a reagent of General Formula I.

FIG. 7 summarizes a method for the preparation of reagents of GeneralFormula VI, wherein group R₁ is a reactive electrophilic or nucleophilicmoiety, wherein group Z is (CH₂)_(n), wherein n is from 1 to 5, andwherein group Z₂ or Z₃ is CH₂CH₂NH—CO(CH₂)₃CONHCH₂Ar, wherein the groupAr represents the aromatic ring to which the spacer Z₂ or Z₃ isappended. A reagent of General Formula I, prepared as described abovewith reference to FIG. 6, is further reacted with an excess of aqueoushydroxylamine, to afford a reagent of General Formula VI.

FIG. 8 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group R is a reactive electrophilicN-hydroxysuccinimidyl ester moiety, wherein group R₃ is selected fromone of an alkyl (e.g., methyl, ethyl, etc.) and a methylene bearing anelectronegative substituent, wherein group Z is (CH₂)₂, and whereingroup Z₂ or Z₃ is CH₂CH₂NHCO(CH₂)₃CONHCH₂Ar, wherein the group Arrepresents the aromatic ring to which the spacer Z₂ or Z₃ is appended.Condensation of the secondary amine intermediate, prepared as describedabove with reference to FIG. 6, with succinic anhydride affords thecorresponding carboxylic acid intermediate, wherein Z is (CH₂)₂.Finally, reaction of the carboxylic acid intermediate with1,3-dicyclohexyl-carbodiimide and N-hydroxysuccinimide affords a reagentof General Formula I, wherein group R is a reactive electrophilicN-hydroxysuccinimidyl ester moiety.

FIG. 9 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group R is a reactive electrophilicN-hydroxysuccinimidyl ester moiety, wherein group R₃ is selected fromone of an alkyl (e.g., methyl, ethyl, etc.) and a methylene bearing anelectronegative substituent, wherein group Z is (CH₂)₃, wherein group Z₂and group Z₃ are each CH₂CONHCH₂Ar, wherein the group Ar represents thearomatic ring to which the spacer Z₂ or Z₃ is appended. Condensation ofiminodiacetic acid, 1,3-dicyclohexylcarbodiimide, andN-hydroxysuccinimide with two equivalents of a reagent selected from oneof methyl, ethyl and cyanomethyl 4-aminomethyl-2-hydroxybenzoate affordsthe secondary amine intermediate, which is isolated as the correspondinghydrochloride salt. To afford different spacers for each of Z₂ and Z₃,one equivalent of a first hydroxybenzoate may be condensed with theiminodiacetic acid followed by condensation with one equivalent of asecond hydroxybenzoate different from the first hydroxybenzoate. Afterneutralization of the hydrochloride salt, the secondary amineintermediate is next condensed with glutaric anhydride to afford thecorresponding carboxylic acid intermediate, wherein Z is (CH₂)₃.Finally, reaction of the carboxylic acid synthetic intermediate with1,3-dicyclohexylcarbodiimide and N-hydroxysuccinimide affords a reagentof General Formula I, wherein group R is a reactive electrophilicN-hydroxysuccinimidyl ester moiety.

FIG. 10 summarizes a method for the preparation of reagents of GeneralFormula VI, wherein group R₁ is a reactive nucleophilic hydrazidemoiety, wherein group Z is (CH₂)₃, and wherein group Z₂ and group Z₃ areeach CH₂CONHCH₂Ar, wherein the group Ar represents the aromatic ring towhich the spacer Z₂ or Z₃ is appended. The iminodiactetic acid adductdepicted in FIG. 10 is first prepared by condensing iminodiacetic acidwith an aliphatic dicarboxylic acid having one terminaltert-butyloxycarbonylhydrazide moiety, to afford the protected hydrazideiminodiacetic acid adduct. Reaction of the iminodiactetic adduct withtwo equivalents of methyl 4-aminomethyl-2-hydroxybenzoate affords thecorresponding diamide intermediate, which is converted into thecorresponding dihydroxamic acid intermediate by reaction with an excessof hydroxylamine. Finally, the protecting group is removed by reactionwith trifluoroacetic acid to afford a reagent of General Formula VI,wherein group R₁ is a reactive nucleophilic hydrazide moiety. To affordalternative spacers for Z₂ and Z₃, one equivalent each of two differenthydroxybenzoate esters may be condensed with the iminodiacetic adduct.

FIG. 11 summarizes a method for the preparation of reagents of GeneralFormula I, wherein group R is a reactive electrophilic or nucleophilicmoiety, wherein group Z is (CH₂)_(n), wherein n is from 1 to 5, whereingroup Z₂ and group Z₃ are each CH₂CONH(CH₂)_(n) ₃ CONHCH₂Ar, wherein n₃is from 1 to 5, and wherein the group Ar represents the aromatic ring towhich the spacer Z₂ or Z₃ is appended. Condensation of iminodiaceticacid; 1,3-dicyclohexylcarbodiimide; and N-hydroxysuccinimide with twoequivalents of a reagent selected from one of methyl, ethyl andcyanomethyl 4-aminomethyl-2-hydroxybenzoate affords the secondary amineintermediate, which is isolated as the corresponding hydrochloride salt.After neutralization of the hydrochloride salt, the secondary amineintermediate is next condensed with a carboxylic acid of the generalformula R(CH₂)_(n) COOH, wherein group R is a reactive electrophilic ornucleophilic moiety, and wherein n is from 1 to 5. The carboxylic acidis first activated by reaction with 1,3-dicyclohexylcarbodiimide andN-hydroxysuccinimide, and affords upon reaction with the secondary amineintermediate a reagent of General Formula I.

EXAMPLES

The following examples present a detailed description of the synthesisof reagents of General Formulas I and VI. Additional examples present adetailed description of the preparation of conjugates of GeneralFormulas III and VII and bioconjugates of General Formulas V and VIII.

Example I Preparation of Ethyl 4-(Bromomethyl)-2-hydroxybenzoic Acid

Ethyl 4-Methyl-2-hydroxybenzoate

4-Methyl-2-hydroxybenzoic acid (20.0 g, 131 mmoles) was dissolved inethanol (300 mL) and concentrated sulfuric acid (2.0 mL) was added. Themixture was refluxed for 40 hours. The volume of the reaction mixturewas reduced to 100 mL, transferred to a separatory funnel, and dilutedwith chloroform (250 mL) and water (200 mL). Solid sodium bicarbonatewas added in small portions until the pH of the aqueous layer was about8 (pH test paper). The mixture in the funnel was shaken well and thelayers separated. The organic layer was washed first with water (150 mL)and then saturated aqueous sodium chloride (150 mL). Finally, theorganic solution was dried over anhydrous magnesium sulfate, filtered,and the solvent evaporated to afford 14.0 g (59% yield) of liquid ethyl4-methyl-2-hydroxybenzoate.

¹H NMR (300 MHz, CHCl₃-d) δ 1.40 (triplet, J=7 Hz, 3H, CH₂CH₃), 2.33(singlet, 3H, ArCH₃), 4.38 (quartet, J=7 Hz, 2H, CH₂CH₃), 6.67 (doublet,J=8 Hz, 1H, ArH), 6.78 (singlet, 1H, ArH), 7.72 (doublet, J=8 Hz, 1H,ArH), 10.81 (singlet, 1H, OH). ¹³C NMR (75 MHz, CHCl₃-d) δ 14.0, 21.7,61.1, 110.1, 117.7, 120.4, 129.7, 146.9, 161.7, 170.3.

Ethyl 4-Bromomethyl-2-hydroxybenzoic Acid

Ethyl 4-methyl-2-hydroxybenzoate (13.1 g, 72.5 mmoles) was dissolved incarbon tetrachloride (150 mL) and N-bromosuccinimide (13.1 g, 73.2mmoles) and benzoyl peroxide (0.2 g, 0.8 mmoles) were added. The mixturewas refluxed for 3.5 hours and then allowed to cool to room temperature.The solids were removed by filtration and the filtrate was evaporated todryness. The crude solid product was crystallized from hexane (100 mL)to afford 5.0 g (27% yield) of ethyl 4-bromomethyl-2-hydroxybenzoic acid(m.p. 64-66° C.).

¹H NMR (300 MHz, CHCl₃-d) δ 1.41 (triplet, J=7 Hz, 3H, CH₂CH₃), 4.40(quartet, J=7 Hz, 2H, CH₂CH₃), 4.40 (singlet, 2H, CH₂Br), 6.90 (doublet,J=8 Hz, 1H, ArH), 6.99 (singlet, 1H, ArH), 7.82 (doublet, J=8 Hz, 1H,ArH), 10.88 (singlet, 1H, OH). ¹³C NMR (75 MHz, CHCl₃-d) δ 14.0, 31.9,61.5, 112.4, 117.9, 119.8, 130.5, 145.5, 161.8, 169.9.

Example II Preparation of Ethyl 4-(Aminomethyl)-2-hydroxybenzoateHydrochloride

Ethyl 4-Aminomethyl-2-hydroxybenzoate Hydrochloride

Ethyl 4-(bromomethyl)-2-hydroxybenzoate (4.8 g, 18.6 mmoles) wasdissolved in dry N,N-di-methylformamide (50 mL) and sodium azide (1.2 g,18.9 mmoles) was added. The suspension was stirred at room temperaturefor 2 hours. The reaction mixture was then diluted with dichloromethane(150 mL) and extracted with 1 N aqueous hydrochloric acid (100 mL),water (100 mL), and saturated aqueous sodium chloride (50 mL). Finally,the solution was then dried over anhydrous magnesium sulfate, filtered,and evaporated to dryness to give ethyl 4-azidomethylsalicyalte as anoil.

Palladium on carbon (0.5 g; 10% [w/w]) was added to a 1 L hydrogenationflask under a nitrogen atmosphere. The crude ethyl4-azidomethyl-2-hydroxybenzoate was dissolved in ethanol (200 mL) andtransferred to the hydrogenation flask. Concentrated aqueoushydrochloric acid (2 mL) was then added, and the flask was affixed tothe Parr hydrogenator. The reaction mixture was shaken under 35 psi ofhydrogen for 4 hours at room temperature. The mixture was then filteredthrough Celite to remove the catalyst, and the filtrate was evaporatedto dryness to afford an off-white solid. Finally, this solid wascrystallized from EtOH to afford 3.1 g (71% yield) of ethyl4-(aminomethyl)-2-hydroxybenzoate hydrochloride (m.p. 240-241° C.).

¹H NMR (300 MHz, DMSO-d₆) δ 1.30 (triplet, J=7 Hz, 3H, CH₂CH₃), 3.99(singlet, 2H, CH₂NH₃), 4.33 (quartet, J=7 Hz, 2H, CH₂CH₃), 7.06(doublet, J=8 Hz, 1H, ArH), 7.15 (singlet, 1H, ArH), 7.77 (doublet, J=8Hz, 1H, ArH), 8.71 (broad singlet, 3H, NH₃), 10.62 (broad singlet, 1H,OH). ¹³C NMR (75 MHz, DMSO-d₆) δ 14.0, 38.7, 41.6, 61.5, 112.9, 117.7,119.8, 130.3, 142.2, 160.2, 168.9.

Example III Preparation of Methyl 4-(Aminomethyl)-2,6-dihydroxybenzoateHydrochloride

Methyl 4-Methyl-2,6-diacetoxybenzoate

Methyl 4-methyl-2,6-dihydroxybenzoate (14.0 grams, 76.8 mmoles) wasdissolved in anhydrous dichloromethane (250 mL) and acetic anhydride (21mL, 223 mmoles) and anhydrous pyridine (18 mL, 223 mmoles) were addedcarefully. The solution was refluxed under dry nitrogen for 30 hours,then cooled to room temperature. The solution was washed twice with 1 Maqueous hydrochloric acid (200 mL portions) and then with saturatedaqueous sodium chloride (200 mL). The dichloromethane solution was driedover anhydrous magnesium sulfate, filtered, and evaporated to anoff-white solid. This crude product was flash chromatographed on silicagel (250 grams) using hexanes:ethyl acetate (70:30 [v/v]) as the elutingsolvent. Fractions containing the major product (R_(f)=0.3) were pooledand evaporated to dryness to afford 19.0 grams (98% yield) of a whitesolid of methyl 4-methyl-2,6-diacetoxybenzoate (m.p. 70-71° C., opencapillary, uncorrected).

¹H NMR (300 MHz, CHCl₃₋d) δ 2.27 (singlet, 6H, COCH₃), 2.37 (singlet,3H, ArCH₃), 3.83 (singlet, 3H, OCH₃), 6.84 (singlet, 2H, ArH). ¹³C NMR(75 MHz, CHCl₃₋d) δ 20.9, 21.5, 52.4, 116.5, 122.1, 143.8, 150.2, 163.9,169.4.

Methyl 4-(Bromomethyl)-2,6-diacetoxybenzoate

Methyl 4-methyl-2,6-diacetoxybenzoate (14.9 grams, 56.0 mmoles) wasdissolved in carbon tetrachloride (200 mL), and N-bromosuccinimide (11.1grams, 62.2 mmoles) and benzoyl peroxide (0.2 grams, 0.8 mmoles) wereadded. The mixture was refluxed under nitrogen. After 3.5 hours, anadditional portion (0.2 grams) of N-bromosuccinimide was added. Refluxwas continued for an additional hour. The reaction mixture was cooled toroom temperature and then in ice for 1 hour, and the solid removed byfiltration. The filtrate was evaporated to dryness. Hexanes (250 mL) wasadded to the residue, and the mixture was boiled until dissolution wasobtained. The hexanes solution was concentrated to about 75 mL when asolid just began to precipitate. The mixture was heated to dissolve thesolid, and the solution was allowed to cool slowly to room temperature.White crystals formed slowly, and the mixture was chilled in ice for 2hours to complete crystallization. The solid was filtered, washed withcold hexanes (100 mL), and dried in vacuo to afford 11.9 grams (62%yield) of methyl 4-bromomethyl-2,6-diacetoxy-benzoate (m.p. 93-95° C.,open capillary, uncorrected). The product was contaminated with a smallamount of methyl 2,6-diacetoxy-4-dibromomethylbenzoate, which did notinterfere with subsequent reactions.

H NMR (300 MHz, CHCl₃-d) δ 2.29 (singlet, 6H, COCH₃), 3.85 (singlet, 3H,OCH₃), 4.41 (singlet, 2H, CH₂), 7.08 (singlet, 2H, ArH). ¹³C NMR (75MHz, CHCl₃-d) δ 20.9, 30.9, 52.7, 119.6, 121.8, 142.4, 150.2, 163.5,169.1.

Methyl 4-(Aminomethyl)-2,6-dihydroxybenzoate Hydrochloride

Methyl 4-(bromomethyl)-2,6-diacetoxybenzoate (45.8 grams, 133 mmoles)was dissolved in N,N-dimethylformamide (150 mL), and sodium azide (8.8g, 135 mmoles) was added. The reaction mixture was stirred for 6 hoursat room temperature. Dichloromethane (350 mL) was added to the reactionmixture, and this solution was washed with water (250 mL), 1 M aqueoushydrochloric acid (250 mL), and saturated aqueous sodium chloride (150mL). The organic solution was dried over anhydrous magnesium sulfate,filtered, and evaporated to a clear, pale yellow oil. The oil wasdissolved in methanol (750 mL) and transferred to a 2 L Parrhydrogenation flask. Palladium on carbon catalyst (10% [w/w], 1.5 g) inwater (10 mL) was added, followed by concentrated hydrochloric acid (15mL). The flask was affixed to a Parr hydrogenator, and the mixture wasshaken at room temperature under 30 psi of hydrogen for 24 hours. Thereaction mixture was filtered through a 0.45 mm nylon filter. Theretained solid was then washed with methanol (150 mL), and concentratedhydrochloric acid (7 mL) was added to the filtrate. The filtrate washeated to reflux for 2 hours, cooled to room temperature, and evaporatedto dryness to give an off-white solid. This solid was dissolved in hotdenatured ethanol (250 mL) and the solution was allowed to cool to roomtemperature. White crystals formed quickly. The mixture was then chilledfor 16-18 hours at 4° C. to complete crystallization. The solid wasfiltered, washed with a little cold ethanol (50 mL) and then diethylether (150 mL), and dried in vacuo over potassium hydroxide pellets toafford 16.0 grams (52% yield based on monobromo starting material) ofmethyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride (m.p.>260° C.,open capillary, uncorrected).

¹H NMR (300 MHz, DMSO-d₆) δ 3.77 (singlet, 3H, OCH₃), 3.83 (singlet, 2H,CH₂), 6.44 (singlet, 2H, ArH), 8.35 (broad singlet, 3H, NH₃), 10.20(singlet, 2H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ 41.9, 52.1, 107.2, 107.3,138.5, 157.6, 168.3.

Example IV Preparation of Ethyl4-[(6-aminohexanoylamino)methyl]-2-hydroxybenzoate Trifluoroacetate

Ethyl(N-tert-Butoxycarbonyl-6-aminohexanoyl)aminomethyl-2-hydroxybenzoate

Ethyl 4-aminomethyl-2-hydroxybenzoate hydrochloride (0.52 g, 1.28mmoles) was suspended in anhydrous N,N-dimethylformamide (25 mL), andN,N-diisopropylethylamine (0.79 mL, 4.53 mmoles) was added, followed byN-tert-butoxycarbonyl-6-aminohexanoic acid succinimidyl ester (0.74 g,2.26 mmoles). The mixture was stirred under dry nitrogen for 18 hours,during which time all solids dissolved. The reaction mixture was dilutedwith ethyl acetate (100 mL) and extracted with 1 N aqueous hydrochloricacid (100 mL). The layers were separated, and the ethyl acetate solutionwas washed with water (100 mL) and saturated aqueous sodium chloride(500 mL). The ethyl acetate solution was dried over anhydrous magnesiumsulfate, filtered, and evaporated to afford an amorphous off-whitesolid. Finally, the solid was crystallized from ethyl acetate, filtered,and dried in vacuo to afford 0.67 g (73% yield) of ethyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2-hydroxybenzoate(m.p. 120-121° C., open capillary, uncorrected).

¹H NMR (300 MHz, DMSO-d₆) δ 1.19 (multiplet, 2H, NHCH₂CH₂CH₂CH₂CH₂CO),1.34 (multiplet, 5H, CH₂CH₂CO and CH₂CH₃), 1.34 (singlet, 9H, C(CH₃)₃),1.49 (multiplet, 2H, NHCH₂CH₂), 2.12 (triplet, J=7 Hz, 2H, CH₂CH₂CO),2.87 (quartet, J=6 Hz, 2H, NHCH₂CH₂), 4.23 (doublet, J=6 Hz, 2H, CH₂Ar),4.32 (quartet, J=7 Hz, CH₂CH₃), 6.74 (triplet, J=6 Hz, 1H, CONHCH₂CH₂),6.80 (doublet, J=8 Hz, 1H, ArH), 6.81 singlet, 1H, ArH), 7.71 (doublet,J=8 Hz, 1H, ArH), 8.34 (triplet, J=6 Hz, 1H, CONHCH₂Ar), 10.58 (singlet,1H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ 14.0, 25.1, 26.3, 28.3, 29.7, 36.3,40.2, 42.9, 61.4, 79.0, 111.6, 116.0, 118.3, 130.4, 146.9, 156.2, 161.9,170.1, 173.0.

Ethyl(6-Aminohexanoyl)aminomethyl-2-hydroxybenzoate Trifluoroacetate

Ethyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2-hydroxybenzoate(0.58 g, 1.41 mmoles) was dissolved in dichloromethane (5 mL) and thesolution was cooled in an ice/water bath. Trifluoroacetic acid (5 mL)was added, and the reaction was allowed to warm to room temperature.After 2 hours, the reaction mixture was evaporated to dryness to givethe product as an oil, which was dried in vacuo over potassium hydroxidepellets to afford 0.59 g (99% yield) of ethyl(6-aminohexanoyl)aminomethyl-2-hydroxybenzoate trifluoroacetate.

¹H NMR (300 MHz, DMSO-d₆) δ 1.28 (multiplet, 5H; H₃CH₂CH₂CH₂CH₂CH₂CO andCH₂CH₃), 1.53 (multiplet, 4H, NH₃CH₂CH₂CH₂CH₂CH₂CO), 2.15 (triplet, J=8Hz, 2H, CH₂CH₂CO), 2.71 (multiplet, 2H, NH₃CH₂CH₂), 4.21 (doublet, J=6Hz, 2H, CH₂Ar), 4.30 (quartet, J=8 Hz, 2H, CH₂CH₃), 6.79 (doublet, J=8Hz, 1H, ArH), 6.81 (singlet, 1H, ArH), 7.68 (doublet, J=8 Hz, 1H, ArH),8.18 (broad singlet, 3H, NH₃), 8.60 (triplet, J=6 Hz, 1H, CONHCH₂Ar),10.58 (broad singlet, 1H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ 14.0, 24.8,25.6, 26.7, 35.1, 38.6, 41.7, 61.3, 111.5, 115.5, 118.3, 130.1, 148.6,160.6, 169.2, 172.6.

Example V Preparation of Methyl4-[(6-Aminohexanoylamino)methyl]-2,6-dihydroxybenzoate Hydrochloride

Methyl(N-tert-Butoxycarbonyl-6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate

Methyl 4-(aminomethyl)-2,6-dihydroxybenzoate hydrochloride (1.50 grams,6.4 mmoles) was suspended in anhydrous N,N-dimethylformamide (25 mL),and N,N-diisopropylethylamine (2.2 mL, 12.8 mmoles) was added, followedby N-tert-butoxycarbonyl-6-aminohexanoic acid succinimidyl ester (2.10grams, 6.4 mmoles). The mixture was stirred under dry nitrogen for 4hours, during which time all solids dissolved. The solvent was thenevaporated to leave a light brown syrup, which was partitioned betweenethyl acetate (70 mL) and 1 M aqueous hydrochloric acid (50 mL). Thelayers were separated, and the ethyl acetate solution was washed withsaturated aqueous sodium bicarbonate (50 mL) and saturated aqueoussodium chloride (50 mL). The ethyl acetate solution was dried overanhydrous sodium sulfate, filtered, and evaporated to an amorphousoff-white solid.

The solid was crystallized from ethyl acetate/hexanes, filtered, anddried in vacuo to afford 2.10 grams (80% yield) of methyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate(m.p. 73-76° C., open capillary, uncorrected).

¹H NMR (300 MHz, DMSO-d₆) δ 1.26 (multiplet, 2H, NHCH₂CH₂CH₂CH₂CH₂CO),1.36 (multiplet, 2H, CH₂CH₂CO), 1.36 (singlet, 9H, C(CH₃)₃), 1.50(multiplet, 2H, NHCH₂CH₂), 2.10 (triplet, J=7 Hz, 2H, CH₂CH₂CO), 2.87(quartet, J=6 Hz, 2H, NHCH₂CH₂), 3.78 (singlet, 3H, OCH₃), 4.08(doublet, J=6 Hz, 2H, CH₂Ar), 6.23 (singlet, 2H, ArH), 6.75 (triplet,J=6 Hz, 1H, CONHCH₂CH₂), 8.26 (triplet, J=6 Hz, 1H, CONHCH₂Ar), 10.04(singlet, 2H, OH). ¹³C NMR (75 MHz, CHCl₃-d) δ 25.0, 26.0, 28.3, 29.3,35.3, 41.7, 52.0, 77.4, 104.3, 105.6, 145.8, 155.8, 158.2, 169.0, 172.4.

Methyl(6-Aminohexanoyl)aminomethyl-2,6-dihydroxybenzoate Hydrochloride

Methyl(N-tert-butoxycarbonyl-6-aminohexanoyl)aminomethyl-2,6-dihydroxy-benzoate(2.00 grams, 4.87 mmoles) was dissolved in ethyl acetate (50 mL), anddry hydrogen chloride was bubbled slowly through the solution. Thereaction mixture warmed as the gas dissolved. After 5 minutes, the gaswas shut off, and the solution was stirred at room temperature. A whiteprecipitate formed in the solution. After 30 minutes, the reactionmixture was chilled in ice for 2 hours, then the solid was filtered,washed with ethyl acetate (50 mL) and then diethyl ether (50 mL), anddried in vacuo over potassium hydroxide pellets to afford 1.68 grams(99% yield) of methyl (6-aminohexanoyl)aminomethyl-2,6-dihydroxybenzoatehydrochloride (m.p. shrinks at 145° C., decomposes with effervescence at152-154° C., open capillary, uncorrected).

¹H NMR (300 MHz, DMSO-d₆) δ 1.29 (multiplet, 2H, H₃CH₂CH₂CH₂CH₂CH₂CO),1.55 (multiplet, 4H, NH₃CH₂CH₂CH₂CH₂CH₂CO), 2.13 (triplet, J=8 Hz, 2H,CH₂CH₂CO), 2.70 (multiplet, 2H, NH₃CH₂CH₂), 3.75 (singlet, 3H, OCH₃),4.07 (doublet, J=6 Hz, 2H, CH₂Ar), 6.26 (singlet, 2H, ArH), 8.05 (broadsinglet, 3H, NH₃), 8.39 (triplet, J=6 Hz, 1H, CONHCH₂Ar), 9.84 (broadsinglet, 2H, OH). ¹³C NMR(75 MHz, CHCl₃-d) δ 24.8, 25.6, 26.7, 35.1,41.8, 52.0, 104.7, 105.5, 145.5, 158.1, 169.0, 172.3.

Example VI Preparation of N-Hydroxysuccinimidyl3-(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)propionate

Methyl 4-Methyl-2-hydroxybenzoate

4-Methyl-2-hydroxybenzoic acid (100 g, 658 mmoles) was dissolved inanhydrous methanol (500 mL) and concentrated sulfuric acid (25 mL) wasadded carefully. The solution was refluxed for 18 hours, then cooled toroom temperature. The reaction mixture was concentrated to about 150 mL,and ethyl acetate (250 mL) was added. The ethyl acetate solution waswashed twice with saturated aqueous sodium bicarbonate (250 mL portions)and then with saturated aqueous sodium chloride (100 mL). The ethylacetate solution was dried over anhydrous sodium sulfate, filtered, andevaporated to a clear, reddish-brown liquid. This crude product wasvacuum distilled (oil pump) to afford a clear, viscous liquid thatsolidified on standing to afford 98.1 g (90% yield) of methyl4-methyl-2-hydroxybenzoate.

¹H NMR (300 MHz, CHCl₃₋d) δ 2.32 (singlet, 3H, ArCH₃), 3.91 (singlet,3H, OCH₃), 6.67 (doublet, J=8 Hz, 1H, ArH), 6.78 (singlet, 1H, ArH),7.69 (doublet, J=8 Hz, 1H, ArH), 10.71 (singlet, 1H, OH). ¹³C NMR (75MHz, CHCl₃₋d) δ 21.8, 52.1, 110.0, 117.9, 120.6, 129.9, 147.3, 161.9,170.9.

Methyl 4-(Bromomethyl)-2-hydroxybenzoate

Methyl 4-methyl-2-hydroxybenzoate (98.1 g, 590 mmoles) was dissolved incarbon tetrachloride (600 mL), and N-bromosuccinimide (105.0 g, 590mmoles) and benzoyl peroxide (0.7 g, 3 mmoles) were added. The mixturewas refluxed under nitrogen. After 2 hours, an additional portion (0.7g) of N-bromosuccinimide was added. Reflux was continued for 16 hours.The reaction mixture was cooled to room temperature and the solidremoved by filtration. The yellow filtrate was evaporated to dryness toafford a thick yellow syrup that solidified on standing. Hexanes (500mL) was added to the solid, and the mixture was boiled until almost allsolids dissolved. The hot hexanes solution was filtered and concentrateduntil a solid just began to precipitate. The mixture was heated todissolve the solid, and the solution was allowed to cool slowly to roomtemperature. Pale yellow crystals formed slowly. The mixture was thenchilled in ice for 2 hours to complete crystallization. Finally, thesolid was filtered, washed with cold hexanes (100 mL), and dried invacuo to afford 83.5 g (58% yield) of methyl4-bromomethyl-2-hydroxybenzoate (m.p. 73-75° C., open capillary,uncorrected).

¹H NMR (300 MHz, CHCl₃₋d) δ 3.95 (singlet, 3H, OCH₃), 4.40 (singlet, 2H,CH₂), 6.90 (doublet, J=8 Hz, 1H, ArH), 7.00 (singlet, 1H, ArH), 7.80(doublet, J=8 Hz, 1H, ArH), 10.78 (singlet, 1H, OH). ¹³C NMR (75 MHz,CHCl₃₋d) δ 32.1, 52.5, 112.4, 118.2, 120.1, 130.7, 145.8, 162.0, 170.5.

Methyl 4-(Azidomethyl)-2-hydroxybenzoate

Methyl 4-(bromomethyl)-2-hydroxybenzoate (83.5 g, 341 mmoles) wasdissolved in dry N,N-dimethylformamide (150 mL) and sodium azide 25.0 g,380 mmoles) was added. The yellow suspension was stirred at roomtemperature, and the solids rapidly dissolved. The solution turnedbrown, and a precipitate of sodium bromide formed. The reaction mixturewas stirred 16 hours, then filtered. The filtrate was evaporated to abrown oil, which was dissolved in a mixture of hexanes and ethyl acetate(1:1 [v/v]. 100 mL). Silica gel (25 g, flash chromatography grade) wasadded to the brown solution, and the mixture was swirled well. Thesilica was removed by filtration on a glass frit, and washed three timeswith hexanes:ethyl acetate (1:1 [v/v], 50 mL portions). The silica gelwas dried on the frit, and the combined filtrates were evaporated todryness to afford a dark yellow liquid. The crude product (which wasutilized for the following reaction) was found to contain some residualN,N-dimethylformamide.

¹H NMR (300 MHz, CHCl₃₋d) δ 3.94 (singlet, 3H, OCH₃), 4.32 (singlet, 2H,CH₂), 6.82 (doublet, J=8 Hz, 1H, ArH), 6.93 (singlet, 1H, ArH), 7.83(doublet, J=8 Hz, 1H, ArH), 10.80 (singlet, 1H, OH). ¹³C NMR (75 MHz,CHCl₃₋d) δ 52.5, 54.3, 112.3, 117.0, 118.7, 130.8, 143.9, 162.1, 170.5.

Methyl 4-(Aminomethyl)-2-hydroxybenzoate Hydrochloride

Crude methyl 4-(azidomethyl)-2-hydroxybenzoate was dissolved in methanol(750 mL) in a 2 L Parr hydrogenation flask. Palladium on carbon catalyst(10% [w/w], 3.8 g) in water (25 mL) was added, followed by concentratedhydrochloric acid (35 mL). The flask was affixed to a Parr hydrogenator,and the mixture was shaken at room temperature under 40 psi of hydrogenfor 16 hours. The reaction mixture was then filtered through a 0.45 mmnylon filter. The retained solid was then washed with methanol (150 mL),water (100 mL), and methanol again (150 mL). The combined filtrates wereevaporated to dryness to afford a tan solid. This solid was dissolved inhot denatured ethanol (150 mL) and the solution was allowed to cool toroom temperature. White crystals formed quickly. Finally, the mixturewas then chilled for 16-18 hours at 4° C. to complete crystallization.The solid was filtered, washed with a little cold ethanol (50 mL) andthen diethyl ether (150 mL), and dried in vacuo over potassium hydroxidepellets to afford 51.5 g (65% yield based on methyl4-bromomethyl-2-hydroxybenzoate) of methyl4-aminomethyl-2-hydroxybenzoate hydrochloride (m.p. 225-227° C., opencapillary, uncorrected).

¹H (300 MHz, DMSO-d₆) δ 3.87 (singlet, 3H, OCH₃), 4.00 (singlet, 2H,CH₂), 7.06 (doublet, J=8 Hz, 1H, ArH), 7.13 (singlet, 1H, ArH), 7.77(doublet, J=8 Hz, 1H, ArH), 8.59 (broad singlet, 3H, NH₃Cl), 10.55(singlet, 1H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ 41.6, 52.6, 113.0, 117.7,119.8, 130.4, 142.1, 160.0, 169.1

Methyl 4-Succinylaminomethyl-2-hydroxybenzoate

Methyl 4-(aminomethyl)-2-hydroxybenzoate hydrochloride (3.00 g, 13.8mmoles) was suspended in dry pyridine (25 mL), andN,N-diisopropylethylamine (2.7 mL, 15.5 mmoles) was added. Thesuspension was stirred in an ice/water bath for 15 minutes, and thensuccinic anhydride (1.36 g, 13.6 mmoles) was added. The mixture wasallowed to warm to room temperature, during which time the startingsolids dissolved. After stirring for 2 hours, the mixture was evaporatedto dryness, and the resulting solid was partitioned between ethylacetate (300 mL) and 1 M aqueous hydrochloric acid (100 mL). The layerswere separated, and the ethyl acetate solution was washed with 1 Mhydrochloric acid (100 mL) and saturated aqueous sodium chloride (100mL). The solution was dried over anhydrous sodium sulfate, filtered, andevaporated to dryness to yield a white solid. Finally, the solid wascrystallized from ethyl acetate/hexanes, filtered, and dried in vacuo toafford 3.02 g (87% yield) of methyl4-succinylaminomethyl-2-hydroxybenzoate (m.p. 161-163° C.).

¹H NMR (300 MHz, DMSO-d₆) δ 2.43 (triplet, J=6 Hz, 2H, CH₂CONH), 2.49(triplet, J=6 Hz, 2H, HO₂CCH₂), 3.87 (singlet, 3H, OCH₃), 4.27 (doublet,J=6 Hz, 2H, ArCH₂NH), 6.83 (doublet, J=8 Hz, 1H, ArH), 6.86 (singlet,1H, ArH), 7.71 (doublet, J=8 Hz, 1H, ArH), 8.43 (triplet, J=6 Hz, 1H,NH), 10.54 (singlet, 1H, OH), 12.12 (singlet, 1H, CO₂H). ¹³C NMR (75MHz, DMSO-d₆) δ 29.1, 30.1, 41.9, 52.5, 111.3, 115.6, 118.4, 130.1,148.7, 160.7, 169.7, 171.7, 174.2.

Methyl 4-Succinylaminomethyl-2-hydroxybenzoate Succinimidyl Ester

Methyl 4-succinylaminomethyl-2-hydroxybenzoate (2.60 g, 10.2 mmoles) wasdissolved in dry tetrahydrofuran (100 mL), and N-hydroxysuccinimide(1.29 g, 11.2 mmoles) and 1,3-di-cyclohexylcarbodiimide (2.10 g, 10.2mmoles) were added. The mixture was stirred at room temperature underdry nitrogen, and the solids rapidly dissolved. After about 20 minutes,a white precipitate formed. The reaction mixture was stirred 16-18hours, then chilled several hours at −20° C. The mixture was filteredcold, and the solid washed with a little tetrahydrofuran (25 mL). Thecombined filtrates were evaporated to dryness, and the residue wascrystallized from ethyl acetate/hexanes, filtered, and dried in vacuo toafford 2.39 g (62% yield) of methyl4-succinyl-aminomethyl-2-hydroxybenzoate succinimidyl ester (m.p.133-135° C.).

¹H NMR (300 MHz, DMSO-d₆) δ 2.56 (triplet, J=7 Hz, 2H, CH₂CONH), 2.80(singlet, 4H, COCH₂CH₂CO), 2.91 (triplet, J=7 Hz, 2H, NO₂CCH₂), 3.87(singlet, 3H, OCH₃), 4.27 (doublet, J=6 Hz, 2H, ArCH₂NH), 6.83 (doublet,J=9 Hz, 1H, ArH), 6.84 (singlet, 1H, ArH), 7.71 (doublet, J=9 Hz, 1H,ArH), 8.51 (triplet, J=6 Hz, 1H, NH), 10.50 (singlet, 1H, OH). ¹³C NMR(75 MHz, DMSO-d₆) δ 25.4, 25.9, 29.2, 41.8, 52.4, 111.4, 115.6, 118.4,130.2, 148.2, 160.4, 168.9, 169.4, 170.2, 170.4.

Example VII Preparation of N-Hydroxysuccinimidyl4-[N-({4-({cyanomethyl)oxycarbonyl]-3-hydroxyphenyl}methyl)carbamoyl]butanoate

Methyl N-tert-Butoxycarbonylaminomethyl-2-hydroxybenzoate

Methyl 4-aminomethyl-2-hydroxybenzoate hydrochloride (10.9 g, 50 mmoles)was suspended in anhydrous methanol (200 mL) anddi-tert-butyldicarbonate (10.9 g, 50 mmoles) and triethylamine (7.0 mL,50 mmoles) were added. The solid rapidly dissolved with the slowevolution of gas. The reaction mixture was stirred at room temperaturefor 18 hours under dry nitrogen, then evaporated to dryness to afford awhite amorphous mass. This mass was partitioned between ethyl acetate(200 mL) and water (100 mL). The layers were separated, and the ethylacetate solution was dried over anhydrous sodium sulfate. The solutionwas filtered and evaporated to a white solid. This solid wascrystallized from ethyl acetate/hexanes, filtered, and dried in vacuo toafford 13.7 g (97% yield) of methylN-tert-butoxycarbonylaminomethyl-2-hydroxybenzoate (m.p. 95-96° C., opencapillary, uncorrected).

¹H NMR (300 MHz, CHCl₃-d) δ 1.42 (singlet, 9H, C(CH₃)₃), 3.90 (singlet,3H, OCH₃), 4.26 (doublet, J=6 Hz, 2H, CH₂Ar), 4.99 (triplet, J=6H, 1H,NH), 6.75 (doublet, J=8 Hz, 1H, ArH), 6.84 (singlet, 1H, ArH), 7.73(doublet, J=8 Hz, 1H, ArH), 10.72 (singlet, 1H, OH). ¹³C NMR (75 MHz,CHCl₃-d) δ 28.5, 44.4, 52.4, 80.0, 111.5, 115.9, 118.2, 130.5, 150.0,156.3, 162.1, 170.8.

N-tert-Butoxycarbonylaminomethyl-2-hydroxybenzoic Acid

Methyl N-tert-butoxycarbonylaminomethyl-2-hydroxybenzoate (8.7 g, 30.9mmoles) was dissolved in dry tetrahydrofuran (100 mL), and potassiumtrimethylsilanolate (4.4 g, 30.9 mmoles, 90% pure) was added. The yellowsolution was refluxed for 24 hours, during which time a tan precipitateformed and the solvent turned light brown. The mixture was evaporated todryness, and the solid was dissolved in cold water (100 mL). The brownsolution was chilled in an ice bath, and saturated aqueous potassiumhydrogen sulfate solution was used to titrate the stirred solution to pH2-3. An off-white solid precipitated during the titration. The solid wasfiltered, washed with cold water, and dried in vacuo over potassiumhydroxide to afford 6.7 g, (81% yield) of crudeN-tert-butoxycarbonylaminomethyl-2-hydroxybenzoic acid (m.p. 141-144°C., decomposes with effervescence on melting, open capillary,uncorrected).

¹H NMR (300 MHz, CHCl₃-d) δ 1.47 (singlet, 9H, C(CH₃)₃), 4.33 (doublet,J=6 Hz, 2H, CH₂Ar), 5.07 (triplet, J=6H, 1H, NH), 6.80 (doublet, J=8 Hz,1H, ArH), 6.88 (singlet, 1H, ArH), 7.81 (doublet, J=8 Hz, 1H, ArH),10.72 (broad singlet, 2H, OH and CO₂H). ¹³C NMR (75 MHz, CHCl₃-d) δ28.5, 44.4, 80.5, 111.1, 115.9, 118.3, 131.5, 148.5, 156.6, 162.6,174.1.

Cyanomethyl N-tert-Butoxycarbonylaminomethyl-2-hydroxybenzoate

N-tert-Butoxycarbonylamino-methyl-2-hydroxybenzoic acid (8.2 g, 30.6mmoles) was suspended in chloroacetonitrile (25 mL), and triethylamine(4.3 mL, 30.6 mmoles) was added. The mixture was stirred under drynitrogen at 50° C., and the solids dissolved. The solution was stirred18 hours, and then cooled to room temperature. The solvent wasevaporated, and the residue was partitioned between ethyl acetate (250mL) and water (250 mL). The layers were separated, and the ethyl acetatelayer was washed with saturated aqueous sodium bicarbonate (100 mL) andsaturated aqueous sodium chloride (100 mL). The solution was dried overanhydrous sodium sulfate, filtered, and evaporated to dryness. Theresidual pale tan solid was dissolved in ethyl acetate (100 mL), andsilica gel (10 g, flash chromatography grade) was added. The mixture wasswirled well and allowed to sit for five minutes at room temperature.The silica was removed by filtration on a glass frit, and washed withethyl acetate (100 mL). The filtrate was evaporated to dryness. Thesolid residue was crystallized from ethyl acetate/hexanes to afford 7.9g (88% yield) of cyanomethylN-tert-butoxycarbonylaminomethyl-2-hydroxybenzoate (m.p. 144-146° C.,open capillary, uncorrected).

¹H NMR (300 MHz, CHCl₃-d) δ 1.45 (singlet, 9H, C(CH₃)₃), 4.30 (doublet,J=6 Hz, 2H, CH₂Ar), 5.00 (singlet, 2H, OCH₂CN), 5.05 (triplet, J=6H, 1H,NH), 6.83 (doublet, J=8 Hz, 1H, ArH), 6.91 (singet, 1H, ArH), 7.77(doublet, J=8 Hz, 1H, ArH), 10.12 (singlet, 1H, OH). ¹³C NMR (75 MHz,CHCl₃-d) δ 28.4, 44.3, 49.0, 80.1, 109.6, 114.2, 116.1, 118.7, 130.5,149.7, 156.2, 162.5, 168.5.

Cyanomethyl 4-Aminomethyl-2-hydroxybenzoate Hydrochloride

Cyanomethyl N-tert-butoxycarbonylaminomethyl-2-hydroxybenzoate (7.7 g,26.2 mmoles) was dissolved in tetrahydrofuran (150 mL), and dry hydrogenchloride was bubbled slowly through the solution. The reaction mixturewarmed as the gas dissolved. After 5 minutes, the gas was shut off, andthe solution was stirred at room temperature. A thick, creamy whiteprecipitate formed in the solution. After 30 minutes, the reactionmixture was chilled in ice for 2 hours, then the solid was filtered,washed with diethyl ether (100 mL) and dried in vacuo over potassiumhydroxide pellets to afford 5.8 g (91% yield) of cyanomethylaminomethyl-2-hydroxybenzoate hydrochloride (m.p. darkens at 210° C.,decomposes at 228° C., open capillary, uncorrected).

¹H NMR (300 MHz, DMSO-d6) δ 4.00 (singlet, 2H, CH₂Ar), 5.20 (singlet,2H, OCH₂CN), 7.05 (doublet, J=8 Hz, 1H, ArH), 7.15 (singlet, 1H, ArH),7.75 (doublet, J=8 Hz, 1H, ArH), 8.62 (broad singlet, 3H, NH₃), 10.38(singlet, 1H, OH). ¹³C NMR (75 MHz, DMSO-d6) δ 41.6, 49.7, 113.0, 116.1,118.0, 119.7, 131.1, 142.3, 159.5, 166.1.

Cyanomethyl 4-Glutarylaminomethyl-2-hydroxybenzoate

Cyanomethyl 4-aminomethyl-2-hydroxybenzoate hydrochloride (1.22 g, 5.0mmoles) was suspended in dry dichloromethane (100 mL), and thesuspension was stirred in an ice/water bath. A solution of glutaricanhydride (0.57 g, 5.0 mmoles) and triethylamine (0.7 mL, 5.0 mmoles) indry dichloromethane (25 mL) was then added dropwise over 15 minutes. Themixture was allowed to warm to room temperature, and the reaction wasstirred for 18 hours. The mixture was evaporated to dryness, and theresulting solid was triturated under cold 0.1 M aqueous hydrochloricacid (50 mL). The solid was collected by filtration, washed with coldwater, and dried in vacuo over potassium hydroxide pellets to afford1.43 g (89% yield) of cyanomethyl4-glutarylaminomethyl-2-hydroxybenzoate (m.p. 125-126° C.).

¹H NMR (300 MHz, DMSO-d₆) δ 1.75 (quintet, J=7 Hz, 2H, CH₂CH₂CH₂), 2.19(triplet, J=7 Hz, 2H, CH₂CONH), 2.22 (triplet, J=7 Hz, 2H, HO₂CCH₂),4.24 (doublet, J=6 Hz, 2H, ArCH₂NH), 5.17 (singlet, 2H, OCH₂CN), 6.81(doublet, J=8 Hz, 1H, ArH), 6.85 (singlet, 1H, ArH), 7.70 (doublet, J=8Hz, 1H, ArH), 8.39 (triplet, J=6 Hz, 1H, NH), 10.5 (very broad singlet,1H, OH), 11.7 (very broad singlet, 1H, CO₂H). ¹³C NMR (75 MHz, DMSO-d₆)δ 20.7, 33.1, 34.5, 41.8, 49.7, 111.2, 115.9, 116.2, 118.5, 130.9,149.1, 160.2, 166.6, 172.3, 174.5.

Cyanomethyl 4-Glutarylaminomethyl-2-hydroxybenzoate Succinimidyl Ester

Cyanomethyl 4-glutarylaminomethyl-2-hydroxybenzoate (1.00 g, 3.1 mmoles)was dissolved in dry tetrahydrofuran (50 mL), and N-hydroxysuccinimide(0.36 g, 3.1 mmoles) and 1,3-dicyclohexylcarbodiimide (0.64 g, 3.1mmoles) were added. The mixture was stirred at room temperature underdry nitrogen, and the solids rapidly dissolved. After about 60 minutes,a white precipitate formed. The reaction mixture was stirred 24 hours,then chilled several hours at −20° C. The mixture was filtered cold, andthe solid washed with a little tetrahydrofuran (10 mL). The combinedfiltrates were evaporated to dryness, and the residue was crystallizedfrom ethyl acetate/hexanes, filtered, and dried in vacuo to afford 1.04g (80% yield) of cyanomethyl 4-glutarylaminomethyl-2-hydroxybenzoatesuccinimidyl ester (m.p. 116-119° C.).

¹H NMR (300 MHz, DMSO-d₆) δ 1.88 (quintet, J=7 Hz, 2H, CH₂CH₂CH₂), 2.30(triplet, J=7 Hz, 2H, CH₂CONH), 2.71 (triplet, J=7 Hz, 2H, NO₂CCH₂),2.80 (singlet, 4H, COCH₂CH₂CO), 4.26 (doublet, J=6 Hz, 2H, ArCH₂NH),5.18 (singlet, 2H, OCH₂CN), 6.82 (doublet, J=8 Hz, 1H, ArH), 6.86(singlet, 1H, ArH), 7.71 (doublet, J=8 Hz, 1H, ArH), 8.44 (triplet, J=6Hz, 1H, NH), 10.18 (broad singlet, 1H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ20.4, 25.5, 29.7, 33.6, 41.8, 49.6, 111.2, 115.9, 116.2, 118.4, 130.9,148.9, 160.1, 166.5, 169.0, 170.5, 171.7.

Example VIII Preparation of N-Hydroxysuccinimidyl4-(N-{[3,5-dihydroxy-4-(methoxy-carbonyl)phenyl]methyl}carbamoyl)butanoate

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate

Glutaric anhydride (2.3 grams, 20 mmoles) was dissolved in drytetrahydrofuran (100 mL), and the suspension was stirred under nitrogen.N,N-diisopropylethylamine (7.0 mL, 40 mmoles) was added, followed bymethyl 4-aminomethyl-2,6-dihydroxybenzoate hydrochloride (4.7 grams, 20mmoles). After stirring for 18 hours, the mixture was evaporated todryness, and the resulting solid was dissolved in dichloromethane (100mL) and allowed to sit at room temperature for 2 hours. Thedichloromethane solution was evaporated to dryness to give a thicksyrup, which was treated with cold 1 M aqueous hydrochloric acid (100mL) for 30 minutes in ice. An off-white solid precipitated, which wasfiltered, washed with cold water, and dried in vacuo to afford 5.5 grams(88% yield) of methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate (m.p.163-164° C.).

¹H (300 MHz, DMSO-d₆) δ 1.74 (quintet, J=8 Hz, 2H, CH₂CH₂CH₂), 2.16(triplet, J=8 Hz, 2H, CH₂CONH), 2.20 (triplet, J=8 Hz, 2H, HO₂CCH₂),3.78 (singlet, 3H, OCH₃), 4.10 (doublet, J=6 Hz, 2H, ArCH₂NH), 6.23(singlet, 2H, ArH), 8.31 (triplet, J=6 Hz, 1H, NH), 10.05 (broadsinglet, 3H, OH and CO₂H). ¹³C NMR (75 MHz, DMSO-d₆) δ 20.7, 33.1, 34.4,41.8, 52.1, 104.4, 105.6, 145.7, 158.2, 169.0, 172.0, 174.5.

Methyl 4-Glutarylaminomethyl-2,6-dihydroxybenzoate Succinimidyl Ester

Methyl 4-glutarylaminomethyl-2,6-dihydroxybenzoate (2.0 grams, 6.4mmoles) was dissolved in dry tetrahydrofuran (80 mL), andN-hydroxysuccinimide (0.8 grams, 7.0 mmoles) and1,3-dicyclohexylcarbodiimide (1.3 grams, 6.4 mmoles) were added. Themixture was stirred at room temperature under dry nitrogen, and thesolids rapidly dissolved. After about 20 minutes, a white precipitateformed. The reaction mixture was stirred 16-18 hours, then chilledseveral hours at −20° C. The mixture was filtered cold, and the solidwashed with a little tetrahydrofuran (25 mL). The combined filtrateswere evaporated to dryness, and the residue was crushed under ice-coldwater (25 mL). The solid was filtered, washed quickly with cold water(25 mL) and then diethyl ether (100 mL), and dried in vacuo. The solidwas recrystallized from ethyl acetate/hexanes to afford 2.5 grams (95%yield) of methyl 4-glutarylaminomethyl-2,6-dihydroxy-benzoatesuccinimidyl ester (m.p. 161-163° C.).

¹H NMR (300 MHz, DMSO-d₆) δ 1.83 (quintet, J=8 Hz, 2H, CH₂CH₂CH₂), 2.26(triplet, J=8 Hz, 2H, CH₂CONH), 2.73 (triplet, J=8 Hz, 2H, NO₂CCH₂),2.80 (singlet, 4H, COCH₂CH₂CO), 3.77 (singlet, 3H, OCH₃), 4.10 (doublet,J=6 Hz, 2H, ArCH₂NH), 6.24 (singlet, 2H, ArH), 8.35 (triplet, J=6 Hz,1H, NH), 10.04 (broad singlet, 2H, OH). ¹³C NMR (75 MHz, DMSO-d₆) δ20.4, 25.4, 29.7, 33.6, 41.8, 52.0, 104.5, 105.6, 145.5, 158.1, 168.9,170.5, 171.4.

Example IX Preparation of Methyl2-Hydroxy-4-[({[3-hydroxy-4-(methoxycarbonyl)-phenyl]methyl}amino)methyl]benzoateHydrochloride

Dissolved methyl 4-aminomethyl-2-hydroxybenzoate hydrochloride (13.07 g,60.1 mmole) and diisopropylethylamine (28.0 mL, 292 mmole) in 225 mL ofN,N-dimethyl-formamide by heating slightly until a solution wasobtained. Allowed the solution to cool to room temperature and addedmethyl 4-bromomethyl-2-hydroxybenzoate (9.81 g, 40.0 mmole). Thereaction was stirred for 1 day at room temperature. The solution wasdiluted with 200 mL of 1N HCl and 250 mL of ethyl acetate and allowed tocool to room temperature. Precipitate was collected by filtration,washed with ethyl acetate and dried in vacuo under a under high vacuum.Obtained 11.32 grams (74% yield) of product as a white solid.

¹H NMR (300 MHz, DMSO-d₆) 3.88 (singlet, 6H, OCH₃), 4.15 (singlet, 4H,NH₂CH₂Ar), 7.08 (doublet, J=8 Hz, 2H, ArH), 7.17 (singlet, 2H, ArH),7.80 (doublet, J=8 Hz, 2H, ArH), 9.72 (broad singlet, 2H, NH₂), 10.53(singlet, 2H, ArH).

Example X Preparation of Methyl4-[((tert-butoxy)-N-{[3-hydroxy-4-(methoxycarbonyl)-phenyl]methyl}carbonylamino)methyl]-2-hydroxybenzoate

Suspended methyl2-hydroxy-4-[({[3-hydroxy-4-(methoxycarbonyl)phenyl]-methyl}amino)methyl]benzoatehydrochloride salt (8.89 g, 23.3 mmole) in 250 mL of methanol and addedtriethylenediamine (6.50 mL, 46.6 mmole). Stirred until the solution washomogenous. Added di-tert-butyldicarbonate and stirred overnight.Removed methanol in vacuo. Passed the material through a large plug ofsilica gel using 30% ethyl acetate in hexane as eluent. Collected thefirst UV active material that eluted from the silica. Removed solventsin vacuo and collected 10.59 grams (100% yield) of the protectedproduct.

¹H NMR (300 MHz, DMSO-d₆) 1.34 (singlet, 9H, C(CH₃)₃), 3.86 (singlet,6H, OCH₃), 4.34 (singlet, 2H, NH₂CH₂Ar), 4.40 (singlet, 2H, NH₂CH₂Ar),6.79 (broad singlet, 4H, ArH), 7.73 (doublet, J=8 Hz, 2H, ArH), 10.50(singlet, 2H, ArH).

Example XI Preparation of Methyl4-[((tert-butoxy)-N-{[4-(methoxycarbonyl)-3-(phenylmethoxy)-phenyl]methyl}carbonylamino)methyl]-2-(phenylmethoxy)benzoate

Dissolved methyl4-[((tert-butoxy)-N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]-methyl}carbonylamino)methyl]-2-hydroxybenzoate(6.86 g, 15.4 mmole) in 125 mL of acetone and added benzyl bromide (3.95mL, 33.2 mmole) followed by potassium carbonate (10.64 g, 76.98 mmole).Heated to reflux and stirred for 16 hours. Filtered away the solids andremoved solvent in vauco. Chromatographed the crude material through alarge column of silica gel and collected 9.44 grams (98% yield) of thebenzyl-protected product.

¹H NMR (300 MHz, DMSO-d₆) 1.35 (singlet, 9H, C(CH₃)₃), 3.75 (singlet,6H, OCH₃), 4.33 (singlet, 2H, NH₂CH₂Ar), 4.41 (singlet, 2H, NH₂CH₂Ar),5.08 (singlet, 4H, ArCH₂OAr), 6.93 (multiplet, 4H, ArH), 7.37(multiplet, 10H, ArH), 7.66 (doublet, J=8 Hz, 2H, ArH).

Example XII Preparation of Methyl4-[({[4-(methoxycarbonyl)-3-(phenylmethoxy)phenyl]-methyl}amino)methyl]-2-(phenylmethoxy)benzoateHydrochloride

Dissolved the methyl4-[((tert-butoxy)-N-{[4-(methoxycarbonyl)-3-(phenyl-methoxy)phenyl]methyl}carbonylamino)methyl]-2-(phenylmethoxy)benzoate(9.44 g, 15.1 mmole) into 150 mL of ethyl acetate and bubbled HCl gasthrough the solution for 15 minutes. Let cool the room temperature andstirred for 1 hour. Removed solvent in vacuo. Dried the oil in vacuounder a high vacuum with NaOH pellets and obtained a crude foam. Thematerial was taken on without further purification.

¹H NMR (300 MHz, DMSO-d₆) 3.78 (singlet, 6H, OCH₃), 4.17 (singlet, 4H,NH₂CH₂Ar), 5.22 (singlet, 4H, ArCH₂OAr), 7.17 (doublet, J=8 Hz, 2H,ArH), 7.48 (multiplet, 14H, ArH), 10.17 (broad singlet, 2H, NH₂).

Example XIII Preparation of Methyl4-[(N-{[4-(methoxycarbonyl)-3-(phenylmethoxy)phenyl]methyl}-6-[(phenylmethoxy)carbonylamino]hexanoylamino)methyl]-2-(phenylmethoxy)benzoate

Dissolved N-Cbz-6-aminohexanoic acid (784 mg, 2.96 mmole) into 10 mL ofN,N-dimethylformamide and cooled to 0° C. Added triethylamine (1.65 mL,11.8 mmole), stirred for 10 minutes, then added isobutylchloroformate(385 uL, 2.68 mmole). Stirred for 45 minutes and then added the crudemethyl4-[({[4-(methoxycarbonyl)-3-(phenylmethoxy)phenyl]methyl}-amino)-methyl]-2-(phenylmethoxy)benzoatehydrochloride from the step above (830 mg, 1.48 mmole). Let warm to roomtemperature and stirred for 3 hours. Diluted the reaction mixture with 1N HCl and extracted with ethyl acetate. Dried the ethyl acetate layerswith brine and anhydrous magnesium sulfate. Filtered, removed solventsin vacuo, and chromatographed on silica gel. Collected 870 mg (76%yield) of product.

¹H NMR (300 MHz, DMSO-d₆) 1.20 (multiplet, 2H, CH₂CH₂CH₂), 1.36(multiplet, 2H, CH₂CH₂CH₂), 1.50 (multiplet, 2H, CH₂CH₂CH₂), 2.28(triplet, J=7.3 Hz, 2H, COCH₂), 2.94 (quartet, J=6.4 Hz, 2H, CONHCH₂),3.75 (singlet, 3H, CO₂CH₃), 3.76 (singlet, 3H, CO₂CH₃), 4.50 (singlet,2H, ArCH₂N), 4.53 (singlet, 2H, ArCH₂N), 4.97 (singlet, 2H, ArCH₂CO₂),5.09 (singlet, 2H, ArCH₂OAr), 5.12 (singlet, 2H, ArCH₂OAr), 6.81(doublet, J=7.9 Hz, 1H, ArH), 6.86 (doublet, J=7.9 Hz, 1H, ArH), 6.91(singlet, 1H, ArH), 7.01 (singlet, 1H, ArH), 7.20 (triplet, J=6.4 Hz,1H, CO₂NH), 7.36 (multiplet, 15H, ArH), 7.64 (doublet, J=7.9 Hz, 1H,ArH), 7.67 (doublet, J=7.9 Hz, 1H, ArH).

Example XIV Preparation of Methyl4-[(6-amino-N-{[3-hydroxy-4-(methoxycarbonyl)-phenyl]methyl}hexanoylamino)methyl]-2-hydroxybenzoate

Dissolved methyl4-[(N-{[4-(methoxycarbonyl)-3-(phenylmethoxy)phenyl]-methyl}-6-[(phenylmethoxy)carbonylamino]hexanoylamino)methyl]-2-(phenylmethoxy)-benzoate(1.20 g, 1.56 mmole) in 400 mL of methanol and then added conc. HCl (400uL) and a small scoop of palladium on carbon (10%). Hydrogenated for 4hours using a Parr shaker at 40 psi. Filtered away the palladium oncarbon and removed the methanol in vacuo. Collected 730 mg (95% yield)of the product as the hydrochloride salt.

¹H NMR (300 MHz, DMSO-d₆) 1.27 (multiplet, 2H, CH₂CH₂CH₂), 1.51(multiplet, 4H, CH₂CH₂CH₂CH₂CH₂), 2.34 (triplet, J=7.3 Hz, 2H, COCH₂),2.71 (multiplet, 2H, NH₃CH₂), 3.87 (singlet, 6H, CO₂CH₃), 4.50 (singlet,2H, ArCH₂N), 4.57 (singlet, 2H, ArCH₂N), 6.76 (multiplet, 4H, ArH), 7.71(doublet, J=7.9 Hz, 1H, ArH), 7.75 (doublet, J=7.9 Hz, 1H, ArH), 7.91(broad singlet, 3H, CH₂NH₃), 10.51 (broad singlet, 2H, ArOH).

Example XV Preparation of Cyanomethyl4-{[2-(3-[(tert-Butoxy)carbonylamino]-N-{[N-({4-[(cyanomethyl)oxycarbonyl]-3-hydroxyphenyl}methyl)carbamoyl]methyl}-propanoylamino)acetylamino]methyl}-2-hydroxybenzoate

To a solution of2-{3-[(tert-butoxy)carbonylamino]-N-(carboxymethyl)-propanoylamino}aceticacid (1.87 g, 6.15 mmole) in 40 mL of N,N-dimethylformamide (DMF) wasadded N-hydroxysuccinimide (1.53 g, 13.3 mmole) and1,3-dicyclo-hexylcarbodiimide (2.66 g, 12.9 mmole). The reaction wasstirred for 8 hours at room temperature. To the solution was addeddisiopropylethylamine (2.70 mL, 28.2 mmole) and cyanomethyl4-aminomethyl-2-hydroxybenzoate hydrochloride (3.14 g, 12.9 mmole), andthe reaction is stirred for 16 hours at room temperature. The solutionhas diluted with ethyl acetate and extracted with 1N HCl and brine. Theorganic phase was dried over anhydrous magnesium sulfate and the solventremoved in vacuo to afford crude product.

Example XVI Preparation of Cyanomethyl4-{[2-(3-Amino-N-{[N-({4-[(cyanomethyl)oxycarbonyl]-3-hydroxyphenyl}methyl)carbamoyl]methyl}propanoylamino)acetylamino]methyl}-2-hydroxybenzoate(9Y-SA(OCH₂CN)-β-Ala-amine)

The crude Cyanomethyl4-{[2-(3-[(tert-Butoxy)carbonylamino]-N-{[N-({4-[(cyanomethyl)oxycarbonyl]-3-hydroxyphenyl}methyl)carbamoyl]methyl}propanoyl-amino)acetylamino]methyl}-2-hydroxybenzoatefrom above was dissolved in 20 mL of dichloroethane and 10 mL oftrifluoroacetic acid (26.0 mmole) was added. The deprotection reactionwas stirred for 2 hours at room temperature. Dichloroethane andtrifluoroacetic acid were removed in vacuo. The resulting oil wasdissolved in 10 mL of ethyl acetate, and the resulting solution wasadded dropwise to 150 mL of rapidly stirred methyl tert-butyl ether. Theproduct precipitated over the course of one hour and was collected byfiltration, washed with methyl tert-butyl ether and dried in vacuo undera high vacuum. Obtained 2.78 grams (65% for two steps) of product as awhite solid.

¹H NMR (300 MHz, DMSO-d₆) 2.64 (triplet, J=7 Hz, 2H, NCH₂CH₂N₂), 2.98(multiplet, 2H, NCH₂CH₂NH₂), 4.03 (singlet, 2H, COCH₂N), 4.19 (singlet,2H, COCH₂N), 4.28 (doublet, J=6 Hz, 2H, ArCH₂NH), 4.32 (doublet, J=6 Hz,2H, ArCH₂NH), 5.19 (singlet, 4H, OCH₂CN), 6.81 (doublet, J=8 Hz, 2H,ArH), 6.86 (singlet, 1H, ArH), 6.89 (singlet, 1H, ArH), 7.72 (multiplet,4H, CH₂NH₂, ArH), 8.75 (triplet, J=6 Hz, 1H, NHCO), 9.14 (triplet, J=6Hz, 1H, NHCO), 10.21 (singlet, 2H, ArOH).

Example XVII Preparation of Methyl4-[(2-{N-[(N-{(3,5-dihydroxy-4-(methoxycarbonyl)phenyl]-methyl}carbamoyl)methyl]-6-[(tert-butoxy)carbonylamino]hexanoylamino}acetyl-amino)methyl]-2,6-dihydroxybenzoate

To a solution of2-{6-[(tert-butoxy)carbonylamino]-N-(carboxymethyl)hexanoyl-amino}aceticacid (1.91 g, 5.51 mmole) in 25 mL of N,N-dimethylformamide (DMF) wasadded N-hydroxysuccinimide (1.31 g, 11.4 mmole) and1,3-dicyclohexylcarbodiimide (2.28 g, 11.1 mmole). The reaction wasstirred for 16 hours at room temperature. A solution containing methyl4-(aminomethyl)-2,6-dihydroxybenzoic acid hydrochloride ((2.58 g, 11.0mmole) and disiopropylethylamine (1.60 mL, 9.19 mmole) in 30 mL of DMFwas added, and the reaction is stirred for 8 hours at room temperature.Solids were removed by filtration, and the solution has diluted withethyl acetate (250 mL) and then extracted with 1N HCl (100 mL) and brine(100 mL). The organic phase was dried over anhydrous magnesium sulfateand the product precipitated by addition of hexane. Obtained 2.41 grams(62% yield) of product.

¹H NMR (300 MHz, DMSO-d6) 1.17 (multiplet, 2H, CH₂), 1.31 (multiplet,2H, CH₂), 1.34 (singlet, 9H, BOC), 1.43 (multiplet, 2H, CH₂), 2.18(triplet, J=7.3 Hz, 2H, CH₂CONR₂), 2.84 (quartet, J=6.0 Hz, 2H,CH₂NHBOC), 3.78 (singlet, 6H, CO₂CH₃), 3.96 (singlet, 2H, NCH₂CO), 4.09(singlet, 2H, NCH₂CO), 4.13 (doublet, J=5.7 Hz, 2H, ArCH₂N), 4.15(doublet, J=5.7 Hz, 2H, ArCH₂N), 6.26 (singlet, 4H, ArH), 6.69 (triplet,J=5.4 Hz, 1H, NHBOC), 8.62 (triplet, J=5.9 Hz, 1H, ArCH₂NH), 9.11(triplet, J=6.0 Hz, 1H, ArCH₂NH), 10.06 (singlet, 4H, ArOH).

Example XVIII Preparation of Methyl4-[(2-{6-Amino-N-[(N-{[3,5-dihydroxy-4-(methoxycarbonyl)-phenyl]methyl}carbamoyl)methyl]hexanoylamino}acetylamino)methyl]-2,6-dihydroxybenzoate(9Y-DHBA(OCH₃)-Hexanoylamine)

The methyl4-[(2-{N-[(N-{(3,5-dihydroxy-4-(methoxycarbonyl)phenyl]methyl}-carbamoyl)methyl]-6-[(tert-butoxy)carbonylamino]hexanoylamino}acetylamino)methyl]-2,6-dihydroxybenzoate(2.31 g, 3.28 mmole) was suspended in 10 mL of dichloroethane and 2 mLof trifluoroacetic acid (26.0 mmole) was added. The deprotectionreaction was stirred for 3 hours at room temperature. Dichloroethane andtrifluoroacetic acid were removed in vacuo, and the resulting residuewas dissolved in 20 mL of methanol, and the product precipitated byaddition of ethyl ether. The product was collected by filtration,waashed with ethyl acetate and dried in vacuo under a high vacuum.Obtained 2.24 grams (97% yield) of product as the trifluoroacetate salt.

¹H NMR (300 MHz, DMSO-d6) 1.22 (multiplet, 2H, CH₂), 1.47 (multiplet,4H, CH₂), 2.20 (triplet, J=7.0 Hz, 2H, CH₂CONR₂), 2.73 (quartet, J=6.7Hz, 2H, CH₂NHBOC), 3.79 (singlet, 6H, CO₂CH₃), 3.96 (singlet, 2H,NCH₂CO), 4.11 (singlet, 2H, NCH₂CO), 4.13 (doublet, J=5.7 Hz, 2H,ArCH₂N), 4.15 (doublet, J=5.7 Hz, 2H, ArCH₂N), 6.27 (singlet, 4H, ArH),7.75 (broad singlet, 3H, NH₃), 8.67 (triplet, J=5.9 Hz, 1H, ArCH₂NH),9.18 (triplet, J=5.9 Hz, 1H, ArCH₂NH), 10.11 (singlet, 4H, ArOH).

Example XIX Preparation of Methyl4-[(2-{4-{N-[(tert-butyloxy)carbonylamino]carbamoyl}-N-[(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)methyl]butanoylamino}-acetylamino)methyl]-2-hydroxybenzoate

To a solution of2-(4-{N-[(tert-butoxy)carbonylamino]carbamoyl}-N-(carboxy-methyl)butanoylamino)aceticacid (1.66 g, 4.59 mmole) in 20 mL of N,N-dimethyl-formamide (DMF) wasadded N-hydroxysuccinimide (1.10 g, 9.56 mmole) and1,3-dicyclohexylcarbodiimide (1.90 g, 9.21 mmole). The reaction wasstirred overnight at room temperature. A solution containing methyl4-aminomethyl-2-hydroxybenzoic acid hydrochloride (2.00 g, 9.19 mmole)and disiopropylethylamine (1.60 mL, 9.19 mmole) in 25 mL of DMF wasadded, and the reaction is stirred for 8 hours at room temperature. Thesolution has diluted with ethyl acetate (250 mL) and then extracted with1N HCl (2×100 mL) and brine (100 mL). The organic phase was dried overanhydrous magnesium sulfate and the product precipitated by addition ofhexane. Obtained 2.00 grams (63% yield) of product as a white powder.

¹H NMR (300 MHz, DMSO-d₆) 1.37 (singlet, 9H, C(CH₃)₃), 1.71 (pentet, J=7Hz, 2H, COCH₂CH₂CH₂CO), 2.07 (triplet, J=7 Hz, 2H, COCH₂CH₂CH₂CO), 2.28(triplet, J=7 Hz, 2H, COCH₂CH₂CH₂CO), 3.86 (singlet, 6H, OCH₃), 4.00(singlet, 2H, COCH₂N), 4.17 (singlet, 2H, COCH₂N), 4.28 (doublet, J=6Hz, 2H, ArCH₂N), 4.32 (doublet, J=6 Hz, 2H, ArCH₂N), 6.81 (doublet, J=8Hz, 2H, ArH), 6.85 (singlet, 2H, ArH), 7.69 (doublet, J=8 Hz, 1H, ArH),7.70 (doublet, J=8 Hz, 1H, ArH), 8.64 (singlet, 1H, NHNHBOC), 8.75(triplet, J=6 Hz, 1H, NHCO), 9.20 (triplet, J=6 Hz, 1H, NHCO), 9.46(singlet, 1H, NHNHBOC), 10.51 (singlet, 2H, ArOH).

Example XX Preparation ofN,N-bis-({N-[(4-(N-hydroxycarbamoyl)-3-hydroxyphenyl)methyl]-carbamoyl}methyl)-N′-[(tert-butoxy)carbonylamino]pentane-1,5-diamide

To 10 mL of water cooled to 0° C. was added hydroxylamine sulfate (1.02g, 6.21 mmole), NaOH (4.75 mL of 6.25 N, 29.9 mmole) and Methyl4-[(2-{4-{N-[(tert-butyloxy)carbonyl-amino]carbamoyl}-N-[(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)methyl]-butanoylamino}acetylamino)methyl]-2-hydroxybenzoate(2.03 g, 2.95 mmole). The stirred solution was allowed to warm to roomtemperature and then stirred for approx. 24 hours. The solution wasextracted with ethyl acetate and the aqueous phase was adjusted to thepH range 5 to 6 by addition of conc. HCl. A precipitate formed whichbecame oily upon standing. The oily material was washed with water andsolidified when dried in vacuo under high vacuum.

¹H NMR (300 MHz, DMSO-d₆) 1.36 (singlet, 9H, C(CH₃)₃), 1.70 (pentet, J=7Hz, 2H, COCH₂CH₂CH₂CO), 2.07 (triplet, J=7 Hz, 2H, COCH₂CH₂CH₂CO), 2.27(triplet, J=7 Hz, 2H, COCH₂CH₂CH₂CO), 3.97 (singlet, 2H, COCH₂N), 4.13(singlet, 2H, COCH₂N), 4.23 (multiplet, 4H, ArCH₂N), 6.67 (doublet, J=8Hz, 2H, ArH), 6.71 (singlet, 2H, ArH), 7.62 (doublet, J=8 Hz, 1H, ArH),7.70 (doublet, J=8 Hz, 1H, ArH), 8.65 (singlet, 1H, NHNHBOC), 8.71(triplet, J=6 Hz, 1H, NHCO), 9.15 (triplet, J=6 Hz, 1H, NHCO), 9.53(singlet, 1H, NHNHBOC), 10.25 (broad singlet, 6H, ArOH, CONHOH).

Example XXI Preparation ofN,N-bis({N-[{4-(N-hydroxycarbamoyl)-3-hydroxyphenyl)-methyl]carbamoyl}methyl)-N′-aminopentane-1,5-diamide

The crudeN,N-bis-({N-[(4-(N-hydroxycarbamoyl)-3-hydroxyphenyl)methyl]-carbamoyl}methyl)-N′-[(tert-butoxy)carbonylamino]pentane-1,5-diamidefrom above was suspended in 20 mL of dichloroethane and 10 mL oftrifluoroacetic acid was added. The deprotection reaction was stirredfor 4 hours at room temperature. Dichloroethane and trifluoroacetic acidwere removed in vacuo, and the resulting residue was dissolved in 10 mLof methanol, which was added dropwise to 150 mL of stirred ethylacetate. A precipitate formed which was collected by filtration, washedwith ethyl acetate and then dried in vacuo under a high vacuum. Obtained1.41 grams (68% yield) of product as a white powder.

¹H NMR (300 MHz, DMSO-d₆) 1.74 (multiplet, 2H, COCH₂CH₂CH₂CO), 2.21(multiplet, 4H, COCH₂CH₂CH₂CO), 3.99 (singlet, 2H, COCH₂N), 4.16(singlet, 2H, COCH₂N), 4.26 (multiplet, 4H, ArCH₂N), 6.67 (doublet, J=8Hz, 2H, ArH), 6.76 (singlet, 2H, ArH), 7.62 (multiplet, 2H, ArH), 8.73(triplet, J=6 Hz, 1H, NHCO), 9.18 (triplet, J=6 Hz, 1H, NHCO), 9.40(broad singlet, 2H, NHNH₂), 11.43 (broad singlet, 2H, ArOH), 12.32(broad singlet, 2H, HONHCO).

Example XXII Preparation of Methyl2-Hydroxy-4-[(4-{N-[2-({2-[4-(N-{(3-hydroxy-4-(methoxy-carbonyl)phenyl]methyl}carbamoyl)butanoylamino]ethyl}dimethylamino)ethyl]-carbamoyl}butanoylamino)methyl]benzoate

NHS4-(N-{(3-hydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)butanoate(6.97 g, 17.8 mmole) was dissolved in 200 mL of tetrahydrofuran (THF)and diethylenetriamine (960 uL, 8.9 mmole) was slowly added. Aprecipitate formed immediately upon addition of diethylenetriamine. Thereaction was stirred for 2 hours at room temperature. Precipitate wascollected by filtration, washed with THF and dried overnight in vacuounder high vacuum. Obtained 7.52 grams of crude product as thecorresponding N-hydroxysuccinimide salt.

¹H NMR (300 MHz, DMSO-d₆) 1.70 (multiplet, 4H, COCH₂CH₂CH₂CO), 2.06(triplet, J=7 Hz, 4H, COCH₂CH₂CH₂CO), 2.14 (triplet, J=7 Hz, 4H,COCH₂CH₂CH₂CO), 2.53 (multiplet, 4H, NHCH₂CH₂NH₂), 2.54 (singlet, 4H,NHS), 3.08 (quartet, J=6 Hz, 4H, NHCH₂CH₂NH₂), 3.86 (singlet, 6H, OCH₃),4.23 (doublet, J=6 Hz, 4H, ArCH₂NH), 6.79 (doublet, J=8 Hz, 2H, ArH),6.81 (singlet, 2H, ArH), 7.70 (doublet, J=8 Hz, 2H, ArH), 7.75 (triplet,J=6 Hz, 2H, CONH), 8.36 (triplet, J=6 Hz, 2H, CONHCH₂Ar). ¹³C NMR (75MHz, DMSO-d₆) 21.5, 25.2, 34.7, 34.8, 41.7, 48.4, 52.4, 52.5, 111.4,115.6, 118.3, 130.3, 148.5, 160.4, 169.4, 172.0, 172.2, 173.7.

Example XXIII Preparation of N-Hydroxysuccinimidyl5-(N,N-bis-{2-[4-(N-{[3-hydroxy-4-(methoxy-carbonyl)phenyl]methyl}carbamoyl)butanoylamino]ethyl}carbamoyl)butanoate

The methyl2-hydroxy-4-[(4-{N-[2-({2-[4-(N-{(3-hydroxy-4-(methoxycarbonyl)-phenyl]methyl}carbamoyl)butanoylamino]ethyl}dimethylamino)ethyl]carbamoyl}-butanoylamino)methyl]benzoate(850 mg, 1.10 mmole) from above was suspended in 150 mL ofN,N-dimethylformamide. Diisopropylethylamine (200 uL, 1.15 mmole)followed by glutaric anhydride (126 mg, 1.10 mmole) were added. Thesuspension was heated and gently stirred until a solution was obtained.The reaction was stirred for 24 hours at room temperature. The volume ofthe solution was reduced to approx. 10 mL by rotary evaporation.N-hydroxysuccinimide (135 mg, 1.17 mmole) and1,3-dicyclohexyl-carbodiimide (230 mg, 1.11 mmole) were added and thereaction was stirred for 24 hours at room temperature. Dicyclohexylureawas removed by filtration. While stirring rapidly, 100 mL of diethylether was added to the filtrate. The white precipitate that formed wascollected by filtration and washed with diethyl ether. The product wasdried in vacuo under high vacuum. Obtained 760 mg (79% yield) of productas a white solid.

¹H NMR (300 MHz, DMSO-d₆) 1.70 (multiplet, 4H, COCH₂CH₂CH₂CO), 1.80(multiplet, 2H, COCH₂CH₂CH₂CO), 2.06 (triplet, J=7 Hz, 4H,COCH₂CH₂CH₂CO), 2.14 (triplet, J=7 Hz, 4H, COCH₂CH₂CH₂CO), 2.40(triplet, J=8 Hz, 2H, COCH₂CH₂CH₂CO), 2.68 (triplet, J=8 Hz, 2H,COCH₂CH₂CH₂CO), 2.79 (singlet, 4H, NHS), 3.16 (multiplet, 4H,NHCH₂CH₂NH₂), 3.27 (multiplet, 4H, NHCH₂CH₂NH₂), 3.86 (singlet, 6H,OCH₃), 4.23 (doublet, J=6 Hz, 4H, ArCH₂NH), 6.78 (doublet, J=8 Hz, 2H,ArH), 6.81 (singlet, 2H, ArH), 7.70 (doublet, J=8 Hz, 2H, ArH), 7.85(triplet, J=6 Hz, 2H, CONH), 7.96 (triplet, J=6 Hz, 2H, CONH), 8.35(triplet, J=6 Hz, 2H, CONHCH₂Ar), 10.49 (singlet, 2H, ArOH).

Example XXIV Preparation of Methyl4-[(4-{N-[2-(4-{N-[(tert-butoxy)carbonylamino]carbamoyl}-N-{2-[4-(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)butanoylamino]-ethyl}butanoylamino)ethyl]carbamoyl}butanoylamino)methyl]-2-hydroxybenzoate

Dissolved NHS5-(N,N-bis-{2-[4-(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]-methyl}carbamoyl)butanoylamino]ethyl}carbamoyl)butanoate(449 mg, 0.517) in 40 mL of tetrahydrofuran. Had to heat slightly toobtain solution. Let cool to room temperature then added tert-butylcarbazate (70 mg, 0.53 mmoL) and stirred for 16 hours. Quenched reactionmixture with 1 N HCl (5 mL) and diluted with ethyl acetate. Extractedwith water and brine. Dried over anhydrous magnesium sulfate, filtered,and removed solvent in vacuo. Collected 380 mg (83%) of crude product.

¹H NMR (300 MHz, DMSO-d₆) 1.37 (singlet, 9H, C(CH₃)₃), 1.65-1.76(multiplet, 6H, COCH₂CH₂CH₂CO), 2.02-2.33 (multiplet, 12H,COCH₂CH₂CH₂CO), 3.15 (multiplet, 4H, NHCH₂CH₂N), 3.29 (multiplet, 4H,NHCH₂CH₂N), 3.86 (singlet, 6H, OCH₃), 4.23 (doublet, J=6 Hz, 4H,ArCH₂NH), 6.78 (doublet, J=8 Hz, 2H, ArH), 6.81 (singlet, 2H, ArH), 7.70(doublet, J=8 Hz, 2H, ArH), 7.85 (triplet, J=6 Hz, 1H, CONH), 7.96(triplet, J=6 Hz, 1H, CONH), 8.35 (triplet, J=6 Hz, 2H, CONHCH₂Ar), 8.64(singlet, 1H, BOCNHNH), 9.47 (singlet, 1H, BOCNHNH), 10.49 (singlet, 2H,ArOH).

Example XXV Preparation ofN,N-bis-[2-(4-{N-[(4-(N-hydroxycarbamoyl)-3-hydroxyphenyl)methyl]-carbamoyl}butanoylamino)ethyl]-N′-[(tert-butoxy)carbonylamino)pentane-1,5-diamide

Dissolved hydroxylamine sulfate (365 mg, 2.22 mmoL) in 10 mL of H₂O andcooled to 0° C. Added 6.25 N NaOH solution (1.80 mL, 11.3 mmoL) followedby methyl 4-[(4-{N-[2-(4-{N-[(tert-butoxy)carbonylamino]carbamoyl}-N-{2-[4-(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]ethyl}carbamoyl)butanoylamino]-ethyl}butanoylamino)-ethyl]carbamoyl}butanoylamino)-methyl]-2-hydroxybenzoate(980 mg, 1.11 mmoL). Let warm to room temperature and stirred for 16hours. Filtered away solids. Cooled to 0° C. and added conc. HCldropwise until a pH of approx. 6 to 7. A precipitate formed initiallybut oiled out after stirring for 20 minutes. Decanted off water, washedwith water, then dried in vacuo under a high vacuum.

¹H NMR (300 MHz, DMSO-d₆) 1.37 (singlet, 9H, C(CH₃)₃), 1.65-1.78(multiplet, 6H, COCH₂CH₂CH₂CO), 2.02-2.18 (multiplet, 10H,COCH₂CH₂CH₂CO), 2.25-2.33 (multiplet, 2H, COCH₂CH₂CH₂CO), 3.13(multiplet, 4H, NHCH₂CH₂N), 3.27 (multiplet, 4H, NHCH₂CH₂N), 4.19(doublet, J=6 Hz, 4H, ArCH₂NH), 6.72 (doublet, J=8 Hz, 2H, ArH), 6.74(singlet, 2H, ArH), 7.61 (doublet, J=8 Hz, 2H, ArH), 7.88 (triplet, J=6Hz, 1H, CONH), 7.97 (triplet, J=6 Hz, 1H, CONH), 8.25 (triplet, J=6 Hz,2H, CONHCH₂Ar), 8.64 (singlet, 1H, BOCNHNH), 9.47 (singlet, 1H,BOCNHNH).

Example XXVI Praparation ofN′-[(4-(N-hydroxycarbamoyl)-3-hydroxyphenyl)methyl]-N-(2-{N-[2-(4-{N-[(N-hydroxycarbamoyl)-3-hydroxyphenyl)methyl]carbamoyl}butanoylamino)ethyl]-4-(N-aminocarbamoyl)butanoylamino}ethyl)pentane-1,5-diamide

Suspended the crudeN,N-bis-[2-(4-{N-[(4-(N-hydroxycarbamoyl)-3-hydroxy-phenyl)methyl]carbamoyl}butanoylamino)ethyl]-N′-[(tert-butoxy)carbonylamino)-pentane-1,5-diamidein 10 mL of dichloroethane and added 2 mL of trifluoroacetic acid(material goes into solution after addition of acid). Stirred at roomtemperature for 3.5 hours. Removed dichloroethane and trifluoroaceticacid in vacuo. Triturated the oil with ethyl acetate, filtered andwashed solid with ethyl acetate. Dried in vauco and collected 590 mg(56% yield for 2 steps) of product.

¹H NMR (300 MHz, DMSO-d₆) 1.65-1.78 (multiplet, 6H, COCH₂CH₂CH₂CO),2.02-2.18 (multiplet, 8H, COCH₂CH₂CH₂CO), 2.21 (multiplet, 2H,COCH₂CH₂CH₂CO), 2.31 (multiplet, 2H, COCH₂CH₂CH₂CO), 3.13 (multiplet,4H, NHCH₂CH₂N), 3.27 (multiplet, 4H, NHCH₂CH₂N), 4.21 (doublet, J=6 Hz,4H, ArCH₂NH), 6.72 (doublet, J=8 Hz, 2H, ArH), 6.74 (singlet, 2H, ArH),7.61 (doublet, J=8 Hz, 2H, ArH), 7.88 (triplet, J=6 Hz, 1H, CONH), 8.02(triplet, J=6 Hz, 1H, CONH), 8.36 (triplet, J=6 Hz, 2H, CONHCH₂Ar), 9.25(broad singlet, 2H), 11.38 (singlet, 2H), 12.28 (broad singlet, 2H).

Preparation of Conjugates of General Formulas III and VII andBioconjugates of General Formulas V and VIII Example XXVII Synthesis of5′-PDBA-Labeled Oligodeoxyribonucleotide Conjugates

Oligodeoxyribonucleotide 7172 (sequence 5′-GATTACGCCAGTTGTACGGAC-3′) wassynthesized on a 1 μmole scale using standard automated phosphoramiditechemistry (Beckman Instruments Oligo 1000 and associated reagents). Aprotected amine-containing phosphoramidite (Aminolink 2, AppliedBiosystems or UniLink Amino Modifier, Clontech) was employed on the sameinstrument to introduce one to four, reactive primary amines onto the5′-end of the oligodeoxyribonucleotide using standard chemistry. Thecompleted oligodeoxyribonucleotide was then cleaved from the support andthe nucleobases deprotected using an UltraFast Deprotection kit (BeckmanInstruments) and the protocol supplied by the manufacturer.

The amino-oligonucleotides were purified by ethanol precipitation,dissolved in 0.8 mL of 0.1 M NaHCO₃, and condensed with of succinimidyl1-carboxamidohexanoyl-3,5-diborylbenzene 1,3-propanediol diester(PDBA-X-NHS) (5 mgs per mmole of primary amino groups on theamino-oligonucleotide in 0.2 mL of anhydrous N,N-dimethylformamide) for2-18 hours at room temperature.

The crude PDBA-modified oligonucleotide was isolated from the reactionmixture by gel filtration on a KwikSep Dextran column (Pierce Chemical)in 0.1 M aqueous triethylammonium acetate, pH 6.5. The PDBA-modifiedoligonucleotide was then concentrated in a vacuum centrifuge to 1 mL,and purified by reverse phase HPLC on a 4.6 mm×250 mm C18 column, with atriethylammonium acetate-acetonitrile gradient. The desired product peakwas collected and evaporated to a small pellet in a vacuum centrifuge,dissolved in 0.5 mL of water, and stored frozen.

Example XXVIII Preparation of Bifunctional 2-Hydroxybenzohydroxamic Acid(Bifunctional-SHA) Magnetic Beads

Ten milliliters of unmodified M280 or M450 magnetic beads (Dynal) weregradually dehydrated into acetonitrile, and converted to aldehydemodified beads using oxalyl chloride activated N,N-dimethylsulfoxide andtriethylamine in dichloromethane at −78° C. The resulting aldehydebearing beads were gradually rehydrated and suspended in 5 mL of 0.1 Msodium acetate pH 5.5. The aldehyde groups were coupledN′-[(4-(N-hydroxycarbamoyl)-3-hydroxy-phenyl)methyl]-N-(2-{N-[2-(4-{N-[(N-hydroxycarbamoyl)-3-hydroxyphenyl)methyl]carbamoyl}-butanoylamino)ethyl]-4-(N-aminocarbamoyl)butanoylamino}ethyl)pentane-1,5-diamide(21Y-SHA-hydrazide) by adding 25 milligrams dissolved in 200 uLN,N-dimethylsulfoxide, and rotating coupling reaction over night at roomtemperature. The beads were then washed extensively with water andstored in 5 mL of 10% ethanol.

Example XXIX Preparation of Bifunctional 2-Hydroxybenzohydroxamic AcidBifunctional-SHA-Sepharose 4B

Bifunctional-SHA-Sepharose 4B was prepared by mixing 130 mg cyanomethyl4-{[2-(3-amino-N-{[N-({4-[(cyanomethyl)oxycarbonyl]-3-hydroxyphenyl}methyl)carbamoyl]methyl}-propanoyl-amino)acetylamino]methyl}-2-hydroxybenzoate(9Y-SA(OCH₂CN)-β-Ala-amine), dissolved in 30 mL 0.2 M NaHCO₃, with. 6.5g HCl washed CNBr activated Sepharose 4B (Pharmacia) overnight at roomtemperature. After the coupling reaction, the gel was washed with waterand suspended in 50 mL 0.5 M NH₂OH, pH 9, and rotated at roomtemperature for two hours. After the conversion reaction, 2 mL 0.5 MTris, pH 8.5 were added and the gel slurry mixed at room temperature for1 hour, and washed with water, 0.5 M NaCl, and water again. Theresulting Bifunctional-SHA-Sepharose 4B was suspended in 30 mL of 20%ethanol, and stored at 4° C.

Example XXX Preparation of Bifunctional 2,6-DihydroxybenzohydroxamicAcid Bifunctional-DHBHA Sepharose 4B

Bifunctional-DHBHA-Sepharose 4B was prepared by mixing 200 milligrams ofmethyl4-[(2-{6-amino-N-[(N-{[3,5-dihydroxy-4-(methoxycarbonyl)phenyl]methyl}-carbamoyl)methyl]hexanoylamino}acetylamino)methyl]-2,6-dihydroxybenzoate(9Y-DHBA(OCH₃)-amine) dissolved in 30 mL 0.2 M NaHCO₃, with 5 g HClwashed CNBr activated Sepharose 4B (Pharmacia), overnight at roomtemperature. After the coupling reaction, the gel was washed with waterand suspended in 50 mL 0.1 M NH₂OH, pH 9, and rotated at roomtemperature for two hours. Finally, the gel was washed with water andsuspended in 30 mL of 20% ethanol, and stored at 4° C.

Example XXXI Preparation of a Phenyldiboronic Acid-α-Biotin AntibodyConjugate

One milliliter of anti-Biotin monoclonal IgG₁ antibody (6.5 mg/mL in 0.1M NaHCO₃) was conjugated with 440 nmoles of PDBA-X-NHS (7.4 ul of 60 mMPDBA-X-NHS dissolved in N,N-dimethylsulfoxide) for 1 hour at roomtemperature. Unconjugated PDBA-X-NHS and its hydrolysis products wereremoved by dialysis. The ultra-violet absorbance spectrum of theresulting conjugate (PDBA-anti-Biotin) exhibited an increase in A₂₆₀relative to A₂₈₀ consistent with phenyldiboronic acid modification.

Example XXXII Preparation of a Phenyldiboronic Acid-Alkaline PhosphataseConjugate

One milliliter of alkaline phosphatase (Sigma, 6 mg/mL) was dialyzedagainst one liter of 0.1 M NaHCO₃, and conjugated with 700 nmoles ofPDBA-X-NHS (10 uL of 70 mM stock in N,N-dimethylformamide) for two hourson ice. Unconjugated PDBA-X-NHS and its hydrolysis products were removedby dialysis in 0.1 M NaHCO₃. The ultra-violet absorbance spectrum of theresulting conjugate (PDBA-AP) exhibited an increase in A₂₆₀ relative toA₂₈₀ consistent with phenylboronic acid modification. The conjugate wasstored at 4° C.

Example XXXIII Preparation of a Bifunctional2,6-Dihydroxybenzohydroxamic Acid Alkaline Phosphatase Conjugate

One milliliter of alkaline phosphatase (Sigma, 6 mg/mL) was dialyzedagainst one liter of 0.1 M NaHCO₃, and conjugated with 714 nmoles ofN-hydroxysuccinimidyl4-(N,N-bis{2-[4-(N-{[3,5-dihydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)butanoylamino]ethyl}carbam-oyl)butanoate(21 Y-DHBA(OCH₃)-NHS) in N,N-dimethylformamide) for two hours on ice.The methyl ester of the conjugate was converted to a hydroxamic acid byadding one milliliter of 2 M NH₂OH, pH 10, and incubating the reactionat 4° C. for six days. The NH₂OH reaction mixture was then dialyzedagainst 0.1 M NaHCO₃ and stored at 4° C.

Example XXXIV Preparation of a Bifunctional 2-HydroxybenzohydroxamicAcid Goat α-Mouse Antibody Conjugate

Two milliliters of goat α-mouse antibody (Rockland, 8.8 mg/mL in 0.1 MNaHCO₃) were conjugated with 2.35 umoles N-Hydroxysuccinimidyl5-(N,N-bis-{2-[4-(N-{[3-hydroxy-4-(methoxycarbonyl)phenyl]methyl}carbamoyl)butanoylamino]-ethyl}carbamoyl)butanoate(21Y-SA(OCH₃)-NHS) for one hour at room temperature. The methyl ester ofthe conjugate was converted to a hydroxamic acid by adding twomilliliters of 2 M NH₂OH, pH 10, adjusting the pH to 10 with 1 N NaOH,and incubating the reaction at room temperature for three days. NH₂OHand unconjugated butanoate (21Y-SA(OCH₃)-NHS) and its hydrolysisproducts were removed by gel filtration on a G-25 Sephadex column(Pharmacia) in 0.1 M NaHCO₃, and the conjugate (SHA-goat α-mouse) wasstored at 4° C.

Example XXXV Preparation of a Bifunctional 2,6-DihydroxybenzohydroxamicAcid Goat α-Mouse Antibody

Two milliliters of goat α-mouse antibody (Rockland, 8.8 mg/mL in 0.1 MNaHCO₃) were conjugated with 2.5 umoles of with (21Y-DHBA(OCH₃)-NHS) for1 hour at room temperature. The methyl ester of the conjugate wasconverted to a hydroxamic acid by adding two milliliters of 2 M NH₂OH,pH 10, adjusting the pH to 10 with 1 N NaOH, and incubating the reactionat room temperature for three days. NH₂OH and unconjugated(21Y-DHBA(OCH₃)-NHS) and its hydrolysis products were removed by gelfiltration on a G-25 Sephadex column (Pharmacia) in 0.1 M NaHCO₃, andthe conjugate (Bifunctional-DHBHA-goat α-mouse) stored at 4° C.

Example XXXVI Polymerase Chain Reaction (PCR) Protocol

A region of Lambda DNA (801 bp) was amplified by the polymerase chainreaction. The PCR reaction contained 200 uM dATP, dCTP, dGTP, and dTTPin addition to Biotin- and PDBA-modified oligonucelotide primers at 1 uMin 1×PCR Buffer II (Perkin Elmer), Lambda DNA (1 ng/uL), and 1U ofThermus aquaticus DNA polymerase. The reaction mixture was denatured at92° C. for one minute and amplified by 35 cycles of PCR at 95° C. for 10seconds, 62° C. for 20 seconds, and 72° C. for 30 seconds, with a finalextension at 72° C. for 5 minutes. The reaction produced 50-100 ng ofamplified product (801 bp), which exhibited retarded mobility relativeto unmodified PCR product during electrophoresis on 1% agarose gels in50 mM Tris, 100 mM borate, 2 mM EDTA, pH 8.3.

Example XXXVII Sandwich Hybridization Detection of Nucleic Acid Probeson Magnetic Particles

A 42-mer oligonucleotide was hybridized with two 21-mer oligonucleotidesbearing 5′-PDBA and Biotin labels in 1.5 M NaCl, 150 mM sodium citrate,pH 7, at 45° C. for ten minutes. Twenty-five microliters of thehybridization mixture was mixed with twentyfive microliters of M280streptavidin-magnetic particles (Dynal) in a polypropylene multiwellplate well. After 30 minutes, the magnetic particles were captured inthe bottom of the well with a magnetic plate, and washed five times with150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween 20, pH 8.

One hundred microliters of Bifunctional-DHBHA-AP (1 ug/mL in 1 mg/mLBSA, 140 mM NaCl, 10 mM Tris-HCl, pH 8) were added to the magneticparticles and mixed well. After 30 minutes, the magnetic particles werecaptured in the bottom of the well with a magnetic plate, and washed sixtimes with 150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween20, pH 8. Alkalinephosphatse substrate (1 mg/mL p-nitrophenylphosphate in 1 Mdiethanolamine buffer, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH 10.4) was added, andincubated at 37° C. for 90 minutes. The Absorbance at 405 nm (A₄₀₅) wasmeasured with an ELISA plate reader (Molecular Devices).

A strong A₄₀₅ was produced when all components of the hybridizationsandwich were present, and the signal was proportional to the amount of42-mer present. Experimental controls lacking either the 42-mer, theBiotin-oligonucleotides and PDBA-oligonucleotides did not produce asignificant A₄₀₅.

Example XXXVIII Sandwich Hybridization Detection of Nucleic Acid Probesin Multiwell Plates

The wells of a polystyrene multiwell plate (Becton Dickinson) werecoated with Bifunctional-DHBHA by filling the wells with 200 uL ofBifunctional-DHBHA-goat α-mouse conjugate (30 ug/mL in 0.1 M NaHCO₃ pH9.0) and incubating overnight at 4° C. The coating solution was removedand the plate backcoated with 5 mg/mL BSA (300 ul per well in 0.2 MNaHCO₃, pH 9.0) for 1 hour at room temperature. The BSA solution wasremoved by washing the plate five times with ELISA Wash Buffer (150 mMNaCl, 20 mM Tris-HCl, 0.02% Tween 20, pH 8.0).

One hundred microliters of unpurified PDBA and biotin labeled PCRproduct were added to 900 ul of 1.5N NaCl, 150 mM sodium citrate, pH 7.0(10×SSC) and serially-diluted in 10×SSC. One hundred microliters of thediluted PCR products were added to the wells and incubated for one hourat room temperature. The plate was then washed five times with ELISAWash Buffer, and 100 ul of Streptavidin-Alkaline Phosphatase (BoehringerMannheim, 0.2 U/mL in 1 mg/ml BSA, 140 mM NaCl, 10 mM Tris-HCl, pH 8.0)were added to each well and incubated for thirty minutes at roomtemperature.

The plate was washed 5 times with ELISA Wash Buffer, and 200 ul ofp-nitrophenyl-phosphate (1 mg/mL in diethanolamine, 1 mM MgCl₂, 0.1 mMZnCl₂, pH 10.4) were added to the plate and incubated at 37° C. for30-60 minutes. Less than 1 uL of PCR product was detected. PCR productlacking either PDBA or biotin labels was not detected.

Example XXXIX Detection of PDBA- & Biotin-Labeled PCR Product onBifunctional-SHA-Magnetic Beads

PDBA- and biotin-labeled PCR product (0.02 μL-5 μL) was diluted into25-100 μL of 1.5 M NaCl, 150 mM sodium citrate, pH 7 (10×SSC), and addedto a polypropylene multiwell plate well containingBifunctional-SHA-magnetic particles (10-50 ul). The particles and PCRproduct were mixed occasionally for 30-60 minutes at room temperature.The magnetic particles were captured in the bottom of the wells with amagnetic plate and washed five times in 150 mM NaCl, 20 mM Tris-HCl,0.02% Tween 20, pH 8 (ELISA Wash Buffer). One hundred microliters ofstreptavidin alkaline-phosphatase (Boehringher Mannheim, 0.2 U/mL in 1mg/mL BSA, NaCl, Tris-HCl, pH 8) were added and mixed with the magneticparticles for 30 minutes at room temperature. The magnetic particleswere captured in the bottom of the wells with a magnetic plate andwashed 5 times with ELISA Wash. Alkaline phosphatase substrate was added(1 m/ml p-nitrophenyl phosphate in 1 M diethanolamine buffer, 1 mMMgCl₂, 0.1 mM ZnCl₂, pH 10.4), and the color developed at 37° C. for10-60 minutes. The lower limit of detection was 50 pg of PCR product.

Example XXXX Detection of a PDBA-Labeled Oligonucleotide Hybridized to aBiotin-Labeled Oligonucleotide

A 42-mer oligonucleotide was hybridized with two 21-mer oligonucleotidesbearing 5′-PDBA and Biotin labels in 1.5 M NaCl, 150 mM sodium citrate,pH 7, at 45 C. for ten minutes. Twenty-five microliters of thehybridization mixture was mixed with 1-50 uL ofBifunctional-SHA-magnetic particles (Dynal, M450) in a polypropylenemultiwell plate well. After 30 minutes, the magnetic particles werecaptured in the bottom of the well with a magnetic plate, and washedfive times with 150 mM NaCl, 20 mM Tris-HCl, 0.02% Tween 20, pH 8.

One hundred microliters of streptavidin-alkalive phosphatase conjugate(SA-AP) (1 ug/mL in 1 mg/mL BSA, 140 mM NaCl, 10 mM Tris-HCl, pH 8) wereadded to the magnetic particles and mixed well. After 30 minutes, themagnetic particles were captured in the bottom of the well with amagnetic plate, and washed six times with 150 mM NaCl, 20 mM Tris-HCl,0.02% Tween 20, pH 8. The particles were mixed with alkaline phosphatsesubstrate (1 mg/mL p-nitrophenyl phosphate in 1 M diethanolamine buffer,1 mM MgCl₂, 0.1 mM ZnCl₂, pH 10.4) and incubated at 37° C. for 90minutes. The A₄₀₅ was measured with a ELISA plate reader (MolecularDevices). As little as 45 pg of oligonucleotide 42-mer was detected.Experimental controls lacking either the 42-mer, or the PDBA or Biotinlabeled oligonucleotides did not produce a significant A₄₀₅.

Example XXXXI Immobilization of a PDBA-anti-Biotin Conjugate onBifunctional-SHA-Sepharose 4B

One mg of PDBA-anti-Biotin, diluted to 1 mL with Tris buffered saline,was applied to small column of Bifunctional-SHA-Sepharose 4B (1.0×2.0cm), and washed extensively with Tris buffered saline. The size of theA₂₈₀ peak of the material not binding to the column indicated thatalmost all of the PDBA-conjugate was immobilized on the column.

Biotin binding activity of the column was assayed by applying to thecolumn 5 mL of 1 ug/mL biotinylated alkaline phosphatase in Trisbuffered saline containing 5 mg/mL bovine serum albumin (BSA). A sampleof the peak of the material flowing through the column was collected forcomparison of the enzymatic activity with a sample of the alkalinephosphatase dilution applied to column. After applying the sample, thecolumn was washed with 20 mL of buffer. After washing, a very smallsample of column material (25 uL liquid containing about 1 uL gel) wascollected to measure the enzymatic activity bound to the gel as a resultof capture by the immobilized anti-biotin antibody.

The alkaline phosphatase activity was measured by incubating 25 uL ofthe enzyme samples in 250 uL of 1 mg/mL p-nitrophenylphosphate in 1 Mdiethanolamine buffer, 1 mM MgCl₂, and 0.1 mM ZnCl₂, pH 10.4, for 20minutes and then adding 650 uL of 0.1 M NaHCO₃, 10 mM EDTA. Relative toa buffer blank, the A₄₀₅ of the sample applied to the column was 1.57,while the A₄₀₅ of the peak of the material not retained by the columnwas only 0.042, indicating that virtually all the enzyme conjugate wascaptured by the column. The small amount of gel assayed produced an A₄₀₅of 1.30, demonstrating that the enzyme was in fact captured by thecolumn.

Example XXXXII Immobilization of a PDBA-Alkaline Phosphatase Conjugateon Bifunctional-SHA-Magnetic Beads

PDBA-conjugated alkaline phosphatase was diluted to 5 ug/mL in Trisbuffered saline containing 5 mg/mL bovine serum albumin. Two hundredmicroliters of diluted PDBA-conjugated enzyme were mixed with 5, 10, or20 uL of Bifunctional-SHA-magnetic beads (Dynal, M280). The enzyme wasalso mixed with 40 uL of unmodified beads as a control. The beads weremixed gently for 10 minutes on ice, after which the beads were capturedwith a rare earth magnet and washed 4 times with Tris buffered saline.The beads were then suspended in 250 uL of 1 mg/mLp-nitrophenylphosphate in 1 M diethanolamine buffer, 1 mM MgCl₂, and 0.1mM ZnCl₂, pH 10.4, and mixed occasionally at 37° C. for 10 minutes. Thereactions were terminated with 750 uL of Tris buffered saline, 5 mMEDTA. The A₄₀₅ relative to a buffer blank was measured to determine thealkaline phosphatase activity bound to the magnetic beads. The controlbeads produced an A₄₀₅ of only 0.15, while the Bifunctional-SHA-magneticbeads produced an A₄₀₅ of 0.62, 0.97, and 1.33 for 5, 10, and 20 uL ofbeads, respectively, indicating the immobilization of significantamounts of PDBA-AP conjugate on the surface of the beads.

Example XXXXIII Capture of a PDBA-Labeled PCR Product Hybridized to aBiotin-Labeled Oligonucleotide

The wells of an amine-coated polystyrene multiwell plate (CorningCostar) were modified with Bifunctional-SHA. The plate was backcoatedwith 5 mg/mL bovine serum albumin (BSA) (300 ul per well in 0.2 MNaHCO₃, pH 9.0) for 1 hour at room temperature. The BSA solution wasremoved by washing the plate five times with ELISA Wash Buffer (150 mMNaCl, 20 mM Tris-HCl, 0.02% Tween 20, pH 8.0).

PDBA-labeled PCR product (2 μL to 10 μL) was serially diluted into 200μL of 1.5 M NaCl, 150 mM sodium citrate, pH 7 (10×SSC), 0.05% Tween20.One-hundred microliter aliquots of the diluted reactions were added tothe multiwell plate. The PDBA-labeled PCR product was hybridized with a5′-biotin labeled 21-mer oligonucleotide for 45 minutes at 50° C. Theplate was then washed five times with ELISA Wash Buffer, and 100 ul ofStreptavidin-Alkaline Phosphatase (Boehringer Mannheim, 0.2 U/mL in 1mg/ml BSA, 140 mM NaCl, 10 mM Tris-HCl, pH 8.0) were added to each welland incubated for thirty minutes at room temperature.

The plate was washed 5 times with ELISA Wash Buffer, and 200 ul ofp-nitrophenyl-phosphate (1 mg/mL in diethanolamine, 1 mM MgCl₂, 0.1 mMZnCl₂, pH 10.4) were added to the plate and incubated at 37° C. for30-60 minutes. Less than 90 ng of hybridized product was detected.Experimental controls lacking either the Biotin-oligonucleotide or thePDBA-PCR product did not produce a significant A₄₀₅.

Example XXXXIV Detection of a PDBA-dUTP PCR Product Hybridized to aBiotin-Labeled Oligonucleotide

PDBA-dUTP labeled PCR product (10 μL) was diluted into 200 μL of 1.5 MNaCl, 150 mM sodium citrate, pH 7 (10×SSC), 0.05% Tween20 containing 100ng of a 5′-biotin labeled 21-mer oligonucleotide, and added to astreptavidin plus coated polystyrene multiwell plate. Hybridized for 60minutes at 50° C., and then washed five times in 150 mM NaCl, 20 mMTris-HCl, 0.02% Tween 20, pH 8 (ELISA Wash Buffer). One hundredmicroliters of Bifunctional-SHA-alkaline phosphatase (1 ug/mL in 1 mg/mLBSA, NaCl, Tris-HCl, pH 8) were added and the plate was incubated for 30minutes at room temperature. Washed the plate 5 times with ELISA Wash.Alkaline phosphatase substrate was added (1 mg/ml p-nitrophenylphosphate in 1M diethanolamine buffer, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH10.4), and the color developed at 37° C. for 10-60 minutes. A strongyellow color developed indicating the detection of significant amountsof immobilized PDBA-dUTP-labeled PCR product. Experimental controlslacking either the incorporated PDBA-dUTP label, or the Biotin-labeledoligonucleotide did not produce a significant A₄₀₅.

Example XXXXV Comparison of Binding of PDBA-Alkaline Phosphatase,PBA-Alkalinephosphatase and PBA-Oxime on modified Bifunctional-SHAMultiwell Plates

The wells of an amine-coated polystyrene multiwell plate (Coming Costar)were modified with Bifunctional-SHA. The plate was backcoated with 5mg/mL bovine serum albumin (BSA) (300 ul per well in 0.2 M NaHCO₃, pH9.0) for 1 hour at room temperature. The BSA solution was removed bywashing the plate five times with ELISA Wash Buffer (150 mM NaCl, 20 mMTris-HCl, 0.02% Tween 20, pH 8.0). The plate was blocked by incubatingwith PBA-oxime (100 ul per well of 10 mM solution in 50 mM Tris, pH 7.5)at room temperature for 30 minutes. The PBA-oxime solution was removedby washing the plate five times with ELISA wash buffer.

PBA- or PDBA-conjugated alkaline phosphatase (100 ul per well of 1 ug/mLin 0.1 M NaHCO₃) was added to the multiwell plate and incubated for 30minutes at room temperature. The plate was then washed five times withELISA Wash Buffer, and 200 ul of p-nitrophenyl-phosphate (1 mg/mL indiethanolamine, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH 10.4) were added to theplate and incubated at 37° C. for 30-60 minutes. Lower A₄₀₅ was observedin wells containing PBA-alkaline phosphatase than PDBA-alkalinephosphatase consistent with more PDBA-alkaline phosphatase being bound.Experimental controls containing only 0.1 M NaHCO₃ or unconjugatedalkaline phosphatase did not produce a significant A₄₀₅.

1. A reagent having the general formula of General Formula I:

wherein group R is an electrophilic or a nucleophilic moiety suitablefor reaction of the reagent with a biologically active species; whereingroup R₂ is selected from one of H and OH moieties; wherein group R₃ isselected from one of an alkyl and a methylene bearing an electronegativesubstituent; wherein group Z is a spacer selected from one of (CH₂)_(n)and CH₂O(CH₂CH₂O)_(n) ₂ , and n is an integer of from 1 to 5, and n₂ isan integer of from 1 to 4; and wherein each of group Z₂ and Z₃ comprise(CH₂)CONH(CH₂)₃CONH(CH₂)₂.
 2. The reagent of claim 1, wherein group Rcomprises one of COOH and a N-hydroxy succinimidyl ester moiety.
 3. Thereagent of claim 1, wherein group Z comprises (CH₂)₃.
 4. The reagent ofclaim 1, wherein group R₂ comprises H.
 5. The reagent of claim 1,wherein group R₃ comprises one of (CH₃) and (CH₂)CN.
 6. The reagent ofclaim 1, wherein group R₂ comprises OH.
 7. The reagent of claim 1,wherein group R comprises COOH; group Z comprises (CH₂)₃; group R₂comprises H; and group R₃ comprises CH₃.
 8. The reagent of claim 1,wherein group R comprises COOH; group Z comprises (CH₂)₃; group R₂comprises H; and group R₃ comprises (CH₂)CN.
 9. The reagent of claim 1,wherein group R comprises a N-hydroxy succinimidyl ester moiety; group Zcomprises (CH₂)₃; group R₂ comprises H; and group R₃ comprises (CH₂)CN.10. The reagent of claim 1, wherein group R comprises COOH; group Zcomprises (CH₂)₃; group R₂ comprises OH; and group R₃ comprises CH₃. 11.A reagent having the general formula of General Formula II:

wherein group R₂ is selected from one of H and OH moieties; whereingroup Z is a spacer selected from one of (CH₂)_(n) and CH₂O(CH₂CH₂O)_(n)₂ , and n is an integer of from 1 to 5, and n₂ is an integer of from 1to 4; wherein each of group Z₂ and group Z₃ comprise a spacer selectedfrom the group consisting of (CH₂)CONH(CH₂)₃CONH(CH₂)₂, and(CH₂)CONH(CH₂); and wherein group R is an electrophilic or anucleophilic moiety suitable for reaction of the reagent with thebiologically active species.
 12. The reagent of claim 11, wherein groupR comprises a hydrazide moiety.
 13. The reagent of claim 11, whereingroup R₂ comprises H.
 14. The reagent of claim 11, wherein group Zcomprises (CH₂)₃.
 15. The reagent of claim 11, wherein each of group Z₂and group Z₃ comprise (CH₂)CONH(CH₂)₃CONH(CH₂)₂.
 16. The reagent ofclaim 11, wherein each of group Z₂ and group Z₃ comprise (CH₂)CONH(CH₂).17. The reagent of claim 11, wherein group R₂ comprises OH.
 18. Thereagent of claim 11, wherein group R comprises a hydrazide moiety; groupR₂ comprises H; group Z comprises (CH₂)₃; and each of group Z₂ and groupZ₃ comprise (CH₂)CONH(CH₂)₃CONH(CH₂)₂.
 19. The reagent of claim 11,wherein group R comprises a hydrazide moiety; group R₂ comprises OH;group Z comprises (CH₂)₃; and each of group Z₂ and group Z₃ comprise(CH₂)CONH(CH₂)₃CONH(CH₂)₂.
 20. The reagent of claim 11, wherein group Rcomprises a hydrazide moiety; group R₂ comprises H; group Z comprises(CH₂)₃, and each of group Z₂ and group Z₃ comprise (CH₂)CONH(CH₂). 21.The reagent of claim 11, wherein group R comprises a hydrazide moiety;group R₂ comprises OH; group Z comprises (CH₂)₃; and each of group Z₂and group Z₃ comprise (CH₂)CONH(CH₂).